Evolution may depend more strongly on variation in gene expression than on differences between variant forms of proteins. Regions of DNA that affect gene expression are highly variable, containing 0.6% polymorphic sites. These naturally occurring polymorphic nucleotides can alter in vivo transcription rates. Thus, one might expect substantial variation in gene expression between individuals. But the natural variation in mRNA expression for a large number of genes has not been measured. Here we report microarray studies addressing the variation in gene expression within and between natural populations of teleost fish of the genus Fundulus. We observed statistically significant differences in expression between individuals within the same population for approximately 18% of 907 genes. Expression typically differed by a factor of 1.5, and often more than 2.0. Differences between populations increased the variation. Much of the variation between populations was a positive function of the variation within populations and thus is most parsimoniously described as random. Some genes showed unexpected patterns of expression--changes unrelated to evolutionary distance. These data suggest that substantial natural variation exists in gene expression and that this quantitative variation is important in evolution.