In the estrogen-primed ovariectomized female rat, the administration of progesterone facilitates sexual receptivity and activates proceptivity for many hours. If the progesterone dose is large (e.g., 2.5 mg), a refractory period follows, during which time animals are less sensitive to additional progesterone. This refractory period has been termed “sequential inhibition” and has been correlated previously with decreased levels of cytosol progestin receptors in the mediobasal hypothalamus-preoptic area (MBH-POA). Our present results indicate that the “sequential inhibition” of mating behavior by progesterone appears to involve a protein synthetic step that is not related causally to decreased levels of cytosol progestin receptors in the MBH-POA. Animals which received subcutaneous injections of the protein synthesis inhibitor, anisomycin (100 mg/kg in saline), 15 min prior to and again 3 hr after progesterone (2.5 mg in propylene glycol) treatment, displayed greater sexual receptivity at 24 hr than did animals which received subcutaneous saline injections. However, cytosol progestin receptor levels in the MBH-POA of animals which received anisomycin plus progesterone were decreased at 24 hr, relative to animals which received saline plus progesterone. Anisomycin did not produce positive or negative behavioral effects at 24 hr when administered 15 min prior to and again 3 hr after propylene glycol, suggesting that the behavioral effects of anisomycin appear to be related to selective blockade of progesterone action. In separate experiments, we administered single subcutaneous injections of anisomycin at various times relative to progesterone (2.5 mg) treatment. According to the time course of effective anisomycin application, the presumed protein synthesis which is responsible for the inhibition of mating behavior occurs between 3 and 13 hr after the administration of progesterone.