A method is described for imaging individual mRNA molecules in fixed cells using multiple singly labeled oligonucleotide probes. Each mRNA species is probed with 48 or more short, singly labeled probes, allowing each mRNA molecule to be visualized as a computationally identifiable fluorescent spot via fluorescence microscopy. The method enables simultaneous detection of three mRNA species in single cells and mRNA detection in yeast, nematodes, fruit fly wing discs, mammalian cell lines, and neurons. As gene expression in individual cells deviates significantly from the average behavior of cell populations, new methods that provide accurate integer counts of mRNA copy numbers in individual cells are needed. Ideally, such methods should also reveal the intracellular locations of the mRNAs, as mRNA localization is often used by cells to spatially restrict the activity of proteins. One candidate for such a method is in situ hybridization followed by microscopic analysis. However, conventional methods using heavily labeled probes suffer from poor sensitivity and spatial resolution. To address these issues, the authors developed a fluorescence in situ hybridization (FISH) procedure that is sensitive enough to detect single mRNA molecules. They used multiple singly labeled probes to detect individual mRNA molecules, which allows for accurate mRNA counts and spatial localization. The method was tested using a doxycycline-controlled gene that produced an mRNA encoding green fluorescent protein (GFP) and possessed 32 tandemly repeated 80 nucleotide-long sequences in its 3'-UTR. The method was also used to detect three different mRNAs in individual cells, demonstrating the ability to simultaneously detect multiple mRNAs. The method was further tested in two commonly studied developmental systems: the nematode, Caenorhabditis elegans, and the fruit fly, Drosophila melanogaster. In the nematode, the method was used to detect mRNA molecules transcribed from the gene alt-2, which is expressed only in the nematode gut. In the fruit fly, the method was used to detect dpp mRNA in wing imaginal discs, demonstrating the method's ability to work in such systems. The method was also tested in Saccharomyces cerevisiae and cultured hippocampal neurons, showing specificity in the behavior of the STL1 gene and β-actin mRNA and Map2 genes, respectively. The method was found to be at least as sensitive as the method of Femino et al. The method's simplicity and accuracy make it a promising tool for genomic-scale studies of mRNA number and localization with applications in systems biology, cell biology, neurobiology, and developmental biology.A method is described for imaging individual mRNA molecules in fixed cells using multiple singly labeled oligonucleotide probes. Each mRNA species is probed with 48 or more short, singly labeled probes, allowing each mRNA molecule to be visualized as a computationally identifiable fluorescent spot via fluorescence microscopy. The method enables simultaneous detection of three mRNA species in single cells and mRNA detection in yeast, nematodes, fruit fly wing discs, mammalian cell lines, and neurons. As gene expression in individual cells deviates significantly from the average behavior of cell populations, new methods that provide accurate integer counts of mRNA copy numbers in individual cells are needed. Ideally, such methods should also reveal the intracellular locations of the mRNAs, as mRNA localization is often used by cells to spatially restrict the activity of proteins. One candidate for such a method is in situ hybridization followed by microscopic analysis. However, conventional methods using heavily labeled probes suffer from poor sensitivity and spatial resolution. To address these issues, the authors developed a fluorescence in situ hybridization (FISH) procedure that is sensitive enough to detect single mRNA molecules. They used multiple singly labeled probes to detect individual mRNA molecules, which allows for accurate mRNA counts and spatial localization. The method was tested using a doxycycline-controlled gene that produced an mRNA encoding green fluorescent protein (GFP) and possessed 32 tandemly repeated 80 nucleotide-long sequences in its 3'-UTR. The method was also used to detect three different mRNAs in individual cells, demonstrating the ability to simultaneously detect multiple mRNAs. The method was further tested in two commonly studied developmental systems: the nematode, Caenorhabditis elegans, and the fruit fly, Drosophila melanogaster. In the nematode, the method was used to detect mRNA molecules transcribed from the gene alt-2, which is expressed only in the nematode gut. In the fruit fly, the method was used to detect dpp mRNA in wing imaginal discs, demonstrating the method's ability to work in such systems. The method was also tested in Saccharomyces cerevisiae and cultured hippocampal neurons, showing specificity in the behavior of the STL1 gene and β-actin mRNA and Map2 genes, respectively. The method was found to be at least as sensitive as the method of Femino et al. The method's simplicity and accuracy make it a promising tool for genomic-scale studies of mRNA number and localization with applications in systems biology, cell biology, neurobiology, and developmental biology.