Female Mexican fruit flies, Anastrepha ludens (Loew), have the capacity to produce more than 1,000 eggs over their lifetime, but fertility of the eggs will depend on the female's capacity to store semen or to replenish semen through remating or both. The two parameters are interrelated in that sexual receptivity depends to a large degree on insemination status. In this controlled study, we measured sperm depletion in singly mated females with continuous access to oviposition substrates by using a squash technique. The spermathecae were sequentially emptied of sperm over the 3 weeks following copulation. Under laboratory conditions, sperm was always found in the ventral receptacle up to 4 weeks following copulation, but was empty in almost all females by the fifth week. These results mirrored previous measurements of egg fertility in singly mated females, which declines at 26–40 d postcopulation.