Abstract Drosophila females engage in multiple matings [1–4] even though they can store sperm in specialized organs for most of their life . The existence of sperm competition in Drosophila has been inferred from the proportion of progeny sired by the second male in double-mating experiments [6–8]. Investigators have used this approach to quantify genetic variation underlying sperm competition [8–10], to elucidate its genetic basis , to identify the dependence of different male competitive ability on the genotype of the females with which they mate  and to discern the potential role of sperm competition in species isolation [13,14]. This approach assumes that the sperm from two males stored in a female compete to fertilize the eggs. The mechanism by which sperm competition is accomplished is still unknown, however. Here, fluorescence microscopy, cytometry, and differently labeled sperm were used to analyze the fate of sperm inside the female's sperm storage organs, to quantify sperm competition, and to assess how closely paternity success corresponds to the appearance and location of the sperm. The results show that the first male's sperm is retained for a shortened period if the female remates, and that the second males that sire more progeny either induce females to store and use more of their sperm or strongly displace resident sperm.