High-resolution electrophoresis has revealed 10 allozymes of esterase-6 (EC 3.1.1.1) in Drosophila melanogaster. The sequences of 13 isolates of the Est6 gene covering all 10 allozymes were obtained and 52 nucleotide differences were found. Sixteen of these cause amino acid replacements, of which three result in charge differences whose size and direction are consistent with the electrophoretic mobilities of the allozymes in which they occur. The smeared electrophoretic phenotype of one allozyme can be explained by the loss of a cysteine residue involved in a disulfide bridge. Several minor mobility variants within the major F and S electrophoretic phenotypes differ by amino acid substitutions that are generally conservative for charge but not for some other properties (size, polarity, or hydrophobicity). Four amino acid differences are found among different isolates of the same allozymes and, overall, 12 amino acid haplotypes occur among the 13 isolates sequenced. Nevertheless, the most common variants within F and S are distinguished by only two amino acids (Asn/Asp at 237 and Thr/Ala at 247), and these are the most likely targets for the selection underlying complementary latitudinal clines in F and S frequencies.