We have characterized a glycosylated, 31 amino-acid peptide of 4932 Da isolated from Drosophila melanogaster males. The mature peptide contains a sugar moiety of 1184 Da at a NDT consensus glycosylation site and a disulfide bond. It is synthesized in the male ejaculatory duct via a 54 amino-acid precursor containing an N-terminal signal peptide and Arg-Lys at the C-terminus which is cleaved off during maturation. The gene contains an intron of 53 bp and is localized in the cytological region 99B of the D. melanogaster genome. The peptide is therefore named DUP99B (for ductus ejaculatorius peptide, cytological localization 99B). The C-terminal parts of mature DUP99B and D. melanogaster sex-peptide (ACP70A) are highly homologous. Injected into virgin females, DUP99B elicits the same postmating responses as sex-peptide (increased oviposition, reduced receptivity). These effects are also induced by de-glycosylated native peptide or synthetic DUP99B lacking the sugar moiety. Presence of the glycosyl group, however, decreases the amount needed to elicit the postmating responses. Homologies in the coding regions of the two exons of DUP99B and sex-peptide, respectively, suggest that the two genes have evolved by gene duplication. Thus, we consider these two genes to be members of the new sex-peptide gene family.