Esterase 6 (Est-6/EST6) is the major beta-carboxylesterase in D. melanogaster and its siblings D. simulans and D. mauritiana. It is expressed in several tissues but its major site of expression is the sperm ejaculatory duct of the adult male. Although EST6 activity affects reproductive fitness, there are high levels of electrophoretic and activity polymorphism, at least within D. melanogaster and D. simulans. Here we present the nucleotide sequences of an Est-6 allele and its flanking regions from each of D. simulans and D. mauritiana and compare them with the published D. melanogaster sequences. As might be expected, replacement sites are significantly less divergent than exon silent sites in all comparisons, suggesting that selection is acting to maintain EST6 structure and function among the three species. Nevertheless, the ratio of the levels of replacement to silent site divergence is still much higher for Est-6 than for seven of ten other genes (including both isozyme-coding loci) for which comparable data have been published for these species. This is consistent with the high levels of EST6 electrophoretic polymorphism within D. melanogaster and D. simulans and implies that selective constraints against amino acid change are relatively weak for EST6. By contrast, comparisons involving promotor sequences show that the level of divergence in the first 350bp 5' of the gene is significantly lower than those for four of the six other loci for which comparable data have been published for these species. In particular, there are two perfectly conserved stretches (-1 to -158bp and -219 to -334bp) each over 100bp long included in this 350bp region. Thus the data suggest a relatively low level of selective constraint on the amino acid sequence of EST6 but a relatively high level of constraint on sequences affecting aspects of its expression.