The ability of cholinergic agents to influence hormone-dependent sexual behavior in female rats was examined. In the first experiment, female sexual behavior, indicated by the incidence of lordosis, was significantly increased in estrogen-treated female rats following bilateral infusion of a cholinergic receptor agonist, carbachol (.5 microgram/cannula), into the medial preoptic area of the brain. Infusion of an artificial cerebrospinal fluid vehicle failed to facilitate lordosis. The incidence of lordosis was normally highest 15 min after carbachol infusion, began to wane by 45 min, and had returned to control levels by 90 min. Further, centrally administered carbachol activated lordosis at lower levels of estrogen priming than did systemically administered progesterone. In a second experiment, female rats, brought into sexual receptivity by administration of estrogen and progesterone, received preoptic infusions of an acetylcholine synthesis inhibitor, hemicholinium-3. Significant reductions in the incidence of lordosis were observed following bilateral infusion of hemicholinium-3 (1.25 microgram/cannula). This inhibition of lordosis was prevented when carbachol (.5 microgram/cannula) was infused along with hemicholinium-3. Results confirm the importance of cholinergic influences on sexual behavior in female rats.