High-throughput gene expression screens provide a quantitative picture of the average expression signature of biological samples. However, the analysis of spatial gene expression patterns with single-cell resolution requires quantitative in situ measurement techniques. Here we describe recent technological advances in RNA fluorescence in situ hybridization (FISH) techniques that facilitate detection of individual fluorescently labeled mRNA molecules of practically any endogenous gene. These methods, which are based on advances in probe design, imaging technology and image processing, enable the absolute measurement of transcript abundance in individual cells with single-molecule resolution.