Seminal fluid proteins from males of many insect species affect the behavior and physiology of their mates. In some cases, these effects result from entry of the proteins into the female's circulatory system. In the fruit fly Drosophila melanogaster, some seminal fluid proteins enter the female's circulatory system after transfer from the male while others remain confined within the reproductive tract. To address where and how seminal fluid proteins enter the hemolymph of the mated female, we compared the kinetics of transfer and localization in mated females of two seminal fluid proteins that enter the hemolymph (Acp26Aa and Acp62F) and one that does not (Acp36DE). We also generated transgenic flies that produce Acp26Aa tagged with Aequorea victoria green fluorescent protein (GFP) to monitor its transfer in vivo. We report that Acps enter the female circulatory system from the posterior vagina immediately after insemination. The ability of Acps to enter the female hemolymph correlates with their ability to cross the intima that lines the posterior vagina. The ventral posterior vagina is structurally unlike other parts of the female reproductive tract in that it lacks muscles. We hypothesize that it has higher permeability thus affording access to the female's circulatory system.