We have determined the nucleotide sequences of seven lacY alleles isolated from natural isolates of Escherichia coli. Nucleotide heterozygosity estimates for this locus were compared to those obtained from previous studies of intraspecific variation at chromosomal loci, revealing that lacY has unusually low synonymous site variation. The average pairwise heterozygosity of synonymous sites (Ks = 0.0112 +/- 0.0100) is the second lowest reported and the lowest for loci that have an equivalent level of nonsynonymous variation. We consider several hypotheses to explain how different forces in evolution could act to create the observed pattern of polymorphism, including selection for translational efficiency and positive selection. Our analysis most strongly supports the hypothesis that positive selection has acted on the lacY locus in E. coli.