Sex and estrous cycle stage affect psychostimulant responses in animals. Cycle stage is typically monitored by vaginal lavage. The present studies tested the hypothesis that vaginal lavage modifies behavioral responses to acute cocaine. Female rats were restrained by briefly holding the tail for either vaginal lavage or touching the thigh, or were undisturbed, for 7-10 days prior to testing. Although habituation to the open-field test chamber was equal in each group, repeated lavage decreased horizontal activity relative to naive rats following acute cocaine (10 mg/kg ip). Lavage and touch attenuated cocaine-stimulated vertical activity. A single lavage prior to testing did not affect cocaine-stimulated motor behavior. Estrous cycle influenced motor activity only in nonlavaged rats. The high cocaine-induced responding observed in proestrous and estrous nonlavaged rats was completely blocked by vaginal lavage. A separate experiment tested the ability of vaginal lavage to establish a conditioned place preference. Vaginal lavage immediately prior to the conditioning session, but neither lavage after conditioning nor touch before, induced a significant preference. These results suggest that vaginal lavage serves as a reinforcing stimulus and interacts with a neural substrate that mediates enhanced locomotor responses to cocaine during proestrus and estrus.