Esterase 6 (EST 6) inDrosophila melanogaster is a male reproductive enzyme transferred to females as a component of the seminal fluid [Richmond, R. C., Gilbert, D. G., Sheehan, K. B., Gromko, M. H., and Butterworth, F. W. (1980).Science 207:1483–1485]. Here we report investigations into the relation between EST 6 and remating by females. EST 6 activity in a strain selected for decreased time to remating is increased over control levels. Inseminated females remated to males carrying null or active alleles show no differences in the timing of remating. However, females inseminated by EST 6-active males remate significantly sooner than females inseminated by null males. Interrupted copulation experiments demonstrate that the remating effect is not due to EST 6 alone but requires other components of the ejaculate. Other evidence suggests that sperm stored in the ventral receptacle respond to EST 6 levels and control remating time. As the first mate of a female who will remate, null-EST 6 males have, under laboratory conditions, a significantly higher fitness than males carrying active alleles. Thus the absence of null alleles of EST 6 in natural populations presents a dilemma suggesting that the remating effect of EST 6 may be balanced by other effects on reproduction.