A wild population of Drosophila paulistorum (Andean semispecies) was analyzed by starch gel electrophoresis for a sex-linked enzyme locus, Tetrazolium oxidase. The results of the analysis of F1 progenies from 106 single female lines from São José do Rio Prêto, Brazil, has allowed us to determine the genotype of 106 wild-collected females and 104 wild-collected males. The population is polymorphic for two alleles with frequencies of 0.56 and 0.44. The observed frequencies among the females of the two homozygotes were 0.23 and 0.13 and of the heterozygote 0.64. The deviation from the frequencies expected according to the Hardy-Weinberg formula is statistically significant (P < 0.005). There is a substantial excess of heterozygotes over the expected number. This observation is interpreted to mean that this polymorphism is maintained by heterosis. This is probably the first reported case of heterosis associated with an enzyme locus in a natural population of Drosophila.