The postmating, prezygotic isolating mechanism known as conspecific sperm precedence (CSP) may play an important role in speciation, and understanding the mechanism of CSP is important in reconstructing its evolution. When a Drosophila simulans female mates with both a D. simulans male and a D. mauritiana male, the vast majority of her progeny are fathered by D. simulans, regardless of the order of mating. The dearth of hybrid progeny does not result from inviability of eggs fertilized by heterospecific sperm or from the relative inviability of heterospecific larvae. Instead, CSP apparently results from a prefertilization obstacle to heterospecific sperm. We identified two independent barriers to heterospecific fertilization, sperm displacement and incapacitation, whose action depends on the order of mating. When a D. simulans female mates first with a conspecific male, the seminal fluid from this mating incapacitates heterospecific sperm transferred two days later. This sperm incapacitation occurs with no change in the retention of stored sperm over time, but does not occur when the conspecific mating lasts for only 5 min. When the order of matings is reversed, the seminal fluid from the second mating physically displaces heterospecific sperm from storage, even if the conspecific copulation lasts only 5 min. Conspecific sperm are not susceptible to displacement by a second conspecific copulation, but are susceptible to interference by heterospecific sperm if the conspecific copulation is interrupted after 12 min. Curing the D. mauritiana males of their infection with the endosymbiont Wolbachia had no effect on CSP. Sperm displacement and incapacitation involve the same basic mechanisms seen in second-male sperm precedence within species, supporting the hypothesis that CSP is an evolutionary by-product of adaptations affecting sperm competition within species.