Over 30 carboxylester hydrolases have been identified in D. melanogaster. Most are classified as acetyl, carboxyl or cholinesterases. Sequence similarities among most of the carboxyl and all the cholinesterases so far characterised from D. melanogaster and other eukaryotes justify recognition of a carboxyl/cholinesterase multigene family. This family shows minimal sequence similarities with other esterases but crystallographic data for a few non-drosophilid enzymes show that the family shares a distinctive overall structure with some other carboxyl and aryl esterases, so they are all put in one superfamily of/beta hydrolases. Fifteen esterase genes have been mapped in D. melanogaster and twelve are clustered at two chromosomal sites. The constitution of each cluster varies across Drosophila species but two carboxyl esterases in one cluster are sufficiently conserved that their homologues can be identified among enzymes conferring insecticide resistance in other Diptera. Sequence differences between two other esterases, the EST6 carboxyl esterase and acetylcholinesterase, have been interpreted against the consensus super-secondary structure for the carboxyl/cholinesterase multigene family; their sequence differences are widely dispersed across the structure and include substantial divergence in substrate binding sites and the active site gorge. This also applies when EST6 is compared across species where differences in its expression indicate a difference in function. However, comparisons within and among species where EST6 expression is conserved show that many aspects of the predicted super-secondary structure are tightly conserved. Two notable exceptions are a pair of polymorphisms in the substrate binding site of the enzyme in D. melanogaster. These polymorphisms are associated with differences in substrate interactions in vitro and demographic data indicate that the alternative forms are not selectively equivalent in vivo.