As in many other insect species, sperm from the second male take precedence over those of the first when Drosophila melanogaster females remate. Two hypotheses have been proposed to account for sperm precedence in this species: (1) first male sperm are released from storage after remating, then restored along with second male sperm: (2) first male sperm remain in storage when females remate and second male sperm are added to them. The primary distinction between these hypotheses is that after remating, sperm are lost from storage in the first, but not in the second. To differentiate between these hypotheses, females initially mated to fertile males were remated within 6 h to XO sterile males, which do not transfer sperm and the subsequent progeny counted. Progeny production decreased significantly as a consequence of remating, indicating that first male sperm are lost from storage when females remate within 6 h. A significant but less pronounced decrease in productivity was also observed when females remated with fertile males. Previous results have indicated that sperm loss does not occur when females wait more than one day to remate, so the phenomenon appears to be limited to a relatively short time after initial mating. © 1990.