Primer3 is a computer program that helps design PCR primers and sequencing probes for various applications, including creating STSs for radiation hybrid mapping and amplifying sequences for SNP discovery. It considers factors such as melting temperature, length, GC content, and secondary structure when selecting primers. Primer3 can be used via its WWW interface or as a software component, with the latter being preferred for high-volume primer selection. The WWW interface is user-friendly and suitable for selecting primers from a few sequences, while primer3_core is more flexible and can be modified for specific needs. Primer3 is written in standard ANSI C and POSIX, ensuring portability and ease of modification. It includes thorough tests to ensure reliability. Primer3's WWW interface allows users to input sequences and select primers, with options to adjust parameters like product size, melting temperature, and mispriming libraries. If no acceptable primers are found, users can adjust constraints to relax requirements. Primer3 is also used in pipelines for tasks like vector clipping, microsatellite identification, and tiling of amplicons across sequences. It is efficient for most users of the WWW interface, but for high-volume applications, factors like library size, primer constraints, and objective function calculations can affect performance. Primer3 is supported by the Howard Hughes Medical Institute and the National Institutes of Health. It is part of a series of primer-picking programs developed at the Whitehead Institute, starting with Primer 0.5. The program has been reviewed and improved by various contributors, and its design draws on earlier versions. Primer3 is available via the WWW and as a software component, with documentation and examples provided for both users and programmers.Primer3 is a computer program that helps design PCR primers and sequencing probes for various applications, including creating STSs for radiation hybrid mapping and amplifying sequences for SNP discovery. It considers factors such as melting temperature, length, GC content, and secondary structure when selecting primers. Primer3 can be used via its WWW interface or as a software component, with the latter being preferred for high-volume primer selection. The WWW interface is user-friendly and suitable for selecting primers from a few sequences, while primer3_core is more flexible and can be modified for specific needs. Primer3 is written in standard ANSI C and POSIX, ensuring portability and ease of modification. It includes thorough tests to ensure reliability. Primer3's WWW interface allows users to input sequences and select primers, with options to adjust parameters like product size, melting temperature, and mispriming libraries. If no acceptable primers are found, users can adjust constraints to relax requirements. Primer3 is also used in pipelines for tasks like vector clipping, microsatellite identification, and tiling of amplicons across sequences. It is efficient for most users of the WWW interface, but for high-volume applications, factors like library size, primer constraints, and objective function calculations can affect performance. Primer3 is supported by the Howard Hughes Medical Institute and the National Institutes of Health. It is part of a series of primer-picking programs developed at the Whitehead Institute, starting with Primer 0.5. The program has been reviewed and improved by various contributors, and its design draws on earlier versions. Primer3 is available via the WWW and as a software component, with documentation and examples provided for both users and programmers.