A molecular approach was employed to detect and clone putative sex-determining loci in dipteran species distantly related to Drosophila. Monoclonal antibodies were available for Sex-lethal (the primary sex-determining gene), as were doublesex (the terminal sex-determining gene) cDNA sequences. Western analysis revealed cross-reacting polypeptides (using anti-SXL) in Calliphora erythrocephala (the bluebottle) whole body extracts were not expressed in adult males or females, although expression was observed in unsexed pupae. Attention was therefore focused on isolating doublesex (dsx) homologues, since initial experiments suggested homologous sequences were present in both Calliphora and Musca domestica (the housefly) genomic DNA. However, despite using a variety of different approaches, we have been unable to isolate a dsx homologue from either of these species. Since neither Calliphora nor Musca appeared to be amenable to cross-hybridisation analysis, a strategy was devised to determine if polypeptides functionally equivalent to DSX proteins were present in Musca. Transcription of the Drosophila yolk protein (yp) genes in the fat body is directly regulated by DSX proteins, such that transcription is activated in females and repressed in males. It has been shown in Calliphora that two yp genes in this species are expressed in an analogous manner, suggesting regulatory proteins (possibly DSX) are likely to be conserved. I report here the cloning of three independent Musca domestica yolk protein gene homologous, and their spatial and temporal expression profiles. Comparisons of dipteran yp gene sequence conservation and the regulation of their expression are made. These results, along with those from ongoing experiments directly related to the newly isolated yp genes described here, suggest the process of sex-determination in Drosophila is unlikely to be a conserved developmental program in dipteran evolution.