Drosophila melanogaster transformed with the esterase 5 (Est-5) gene from Drosophila pseudoobscura were used to assess the evolutionary basis for differences in the sex- and tissue-specific expression of the esterase 5 (EST 5) enzyme in D. pseudoobscura relative to its homologue in D. melanogaster, EST 6. EST 5 is expressed in the eyes and hemolymph of transformed D. melanogaster just as it is in D. pseudoobscura, but it is not detectable in the ejaculatory duct, where the homologous enzyme, EST 6, is most abundant. EST 5 also occurs at equal levels in both sexes of the transformants and D. pseudoobscura, whereas EST 6 is more abundant in male than in female D. melanogaster. Northern analysis of transformed and untransformed flies indicates that the expression patterns of EST 5 and EST 6 are controlled at the level of transcription and suggests that regulatory differences between Est-6 and Est-5 have evolved mainly through cis-acting regulatory changes in the two loci rather than through alterations in trans-acting factors. Equal expression of EST 5 in male and female transformants also indicates that the X-chromosome-linked Est-5 gene of D. pseudoobscura, when isolated as a 4.5-kilobase restriction fragment, is not dosage compensated after integration into an autosome of D. melanogaster.