Mating triggers behavioral and physiological changes in the Drosophila melanogaster female, including an elevation of egg laying. Seminal fluid molecules from the male accessory gland are responsible for initial behavioral changes, but persistence of these changes requires stored sperm. Using genetic analysis, we have identified a seminal fluid protein that is responsible for an initial elevation of egg laying. This molecule, Acp26Aa, has structural features of a prohormone and contains a region with amino acid similarity to the egg-laying hormone of Aplysia. Acp26Aa is transferred to the female during mating, where it undergoes processing. Here we report the generation and analysis of mutants, including a null, in Acp26Aa. Females mated to male flies that lack Acp26Aa lay fewer eggs than do mates of normal males. This effect is apparent only on the first day after mating. The null mutation has no other detectable physiological or behavioral effects on the male or the mated female.