Light-generated oligonucleotide arrays enable rapid DNA sequence analysis through photolithographic techniques. These arrays, or DNA chips, allow for parallel DNA hybridization analysis, directly yielding sequence information. A 1.28 × 1.28 cm array of 256 different octanucleotides was produced in 16 chemical reaction cycles, taking 4 hours. Fluorescently labeled oligonucleotide targets were detected using epifluorescence microscopy, showing strong fluorescence signals from complementary probes, demonstrating sequence specificity. Sequencing by hybridization (SBH) uses short oligonucleotide probes to identify complementary sequences on a longer DNA target. Hybridization patterns reconstruct the target DNA sequence. SBH can be implemented by attaching probes to a surface in an array format, allowing direct identification of complementary sequences. A 256-octanucleotide array was generated using a solution-channeling device, enabling high-density arrays. Light-directed synthesis of oligonucleotide probes is achieved through photolabile protecting groups, surface linker chemistry, and combinatorial synthesis strategies. A matrix of 256 spatially defined oligonucleotide probes was generated, and the ability to identify complementary sequences was demonstrated by hybridizing fluorescently labeled octanucleotides. The hybridization pattern showed high base specificity and revealed the sequence of oligonucleotide targets. The synthesis process involves photodeprotection and coupling cycles, with photolithography enabling miniaturization of high-density arrays. The photolabile 5'-hydroxyl protecting groups were designed to avoid undesirable nucleoside photochemistry and ensure equal deprotection in different illuminated sites. The synthesis support was prepared with a glass substrate, derivatized with a solution of bis(2-hydroxyethyl)aminopropyltriethoxysilane. The chemical coupling efficiencies were measured using various methods, including hexaethylene glycol derivatized control pore glass and direct coupling on the glass synthesis supports. The coupling efficiencies ranged between 85% and 98%. The results showed that the probe matrix could be efficiently generated by light-directed synthesis, with the ability to determine the identity of DNA target sequences. The array of all tetranucleotides was produced in 16 cycles, taking 4 hours. Combinatorial strategies allow for exponential increases in the number of compounds while linear increases in chemical coupling cycles. The potential of light-directed combinatorial synthesis was demonstrated by the synthesis of a probe matrix containing all possible tetranucleotides. The arrays have broad applications in genetic diagnostics, pathogen detection, and DNA molecular recognition. They can also be used to study sequence specificity of RNA- or protein-DNA interactions. The high information content of light-directed oligonucleotide arrays will change genetic diagnostic testing, enabling simultaneous sequence comparisons of hundreds to thousands of genes. Custom arrays can be constructed to contain genetic markers for rapid identification ofLight-generated oligonucleotide arrays enable rapid DNA sequence analysis through photolithographic techniques. These arrays, or DNA chips, allow for parallel DNA hybridization analysis, directly yielding sequence information. A 1.28 × 1.28 cm array of 256 different octanucleotides was produced in 16 chemical reaction cycles, taking 4 hours. Fluorescently labeled oligonucleotide targets were detected using epifluorescence microscopy, showing strong fluorescence signals from complementary probes, demonstrating sequence specificity. Sequencing by hybridization (SBH) uses short oligonucleotide probes to identify complementary sequences on a longer DNA target. Hybridization patterns reconstruct the target DNA sequence. SBH can be implemented by attaching probes to a surface in an array format, allowing direct identification of complementary sequences. A 256-octanucleotide array was generated using a solution-channeling device, enabling high-density arrays. Light-directed synthesis of oligonucleotide probes is achieved through photolabile protecting groups, surface linker chemistry, and combinatorial synthesis strategies. A matrix of 256 spatially defined oligonucleotide probes was generated, and the ability to identify complementary sequences was demonstrated by hybridizing fluorescently labeled octanucleotides. The hybridization pattern showed high base specificity and revealed the sequence of oligonucleotide targets. The synthesis process involves photodeprotection and coupling cycles, with photolithography enabling miniaturization of high-density arrays. The photolabile 5'-hydroxyl protecting groups were designed to avoid undesirable nucleoside photochemistry and ensure equal deprotection in different illuminated sites. The synthesis support was prepared with a glass substrate, derivatized with a solution of bis(2-hydroxyethyl)aminopropyltriethoxysilane. The chemical coupling efficiencies were measured using various methods, including hexaethylene glycol derivatized control pore glass and direct coupling on the glass synthesis supports. The coupling efficiencies ranged between 85% and 98%. The results showed that the probe matrix could be efficiently generated by light-directed synthesis, with the ability to determine the identity of DNA target sequences. The array of all tetranucleotides was produced in 16 cycles, taking 4 hours. Combinatorial strategies allow for exponential increases in the number of compounds while linear increases in chemical coupling cycles. The potential of light-directed combinatorial synthesis was demonstrated by the synthesis of a probe matrix containing all possible tetranucleotides. The arrays have broad applications in genetic diagnostics, pathogen detection, and DNA molecular recognition. They can also be used to study sequence specificity of RNA- or protein-DNA interactions. The high information content of light-directed oligonucleotide arrays will change genetic diagnostic testing, enabling simultaneous sequence comparisons of hundreds to thousands of genes. Custom arrays can be constructed to contain genetic markers for rapid identification of