Sequence variation was studied in a 2.2-kb region encompassing the esterase-5B locus in Drosophila pseudoobscura from two California populations. In these populations, two common electrophoretic classes and many less frequent variants occur, and it was formerly shown by KEITH (1983) that allele frequencies differed from random distribution under an infinite allele model. Nucleotide polymorphisms were determined in 16 sequences representing 14 electrophoretic classes. There was no significant sequence differentiation between populations, and both synonymous and nonsynonymous polymorphisms are distributed homogeneously along the sequence. The data show that the two major electrophoretic classes are heterogeneous at the amino acid level with no diagnostic amino acid(s) distinguishing them. At the nucleotide level, members of one major class are more similar to members of other electrophoretic classes than they are to each other. It appears that random combinations of the neutral amino acid polymorphisms and other undefined physical properties of the proteins generate the different electrophoretic classes and maintain considerable variation at Est-5B.