Esterase 6, a component of the seminal fluid of Drosophila melanogaster males, hydrolyzes cis-vaccenyl acetate, a lipid made only by males, to cis-vaccenyl alcohol. This reaction occurs in the female reproductive tract and is virtually complete within 6 hours after copulation. Both the alcohol and the acetate decrease the number of matings among pairs of virgin flies in which the female is treated topically with these substances. Although females tested 10 minutes after copulation elicit less courtship than virgin females, females tested 6 hours after copulation stimulate even less courtship if they received active esterase 6 in the seminal fluid of their respective mates. Either the alcohol or a derivative appears to be an antiaphrodisiac that decreases courtship elicited by inseminated females and thus reduces the probability of further mating. Thus the activity of the pheromone depends on a final reaction which occurs in the female, using both substrate and enzyme provided by the male.