Early life experience impacts emotional development in the infant. In rat pups, repeated, brief (i.e., 15 min) maternal separation (MS15) during the first 1-2 postnatal weeks has been shown to increase active maternal care and to reduce later anxiety-like behavior in the offspring. We hypothesized that the anxiolytic effect of MS15 is partly due to increased intestinal release of cholecystokinin (CCK) in rat pups as a result of increased maternal contact. We predicted that rats with a history of MS15 would display less anxiety in the elevated plus maze (EPMZ) and novelty-suppressed feeding (NSF) tests, as compared with nonseparated (NS) controls, and that the anxiolytic effect of MS15 would be attenuated in rats in which daily MS15 was accompanied by systemic administration of a CCK-1 receptor antagonist (i.e., devazepide). Treatment groups included NS control litters, litters exposed to MS15 from postnatal days (P)1-10, inclusive, and litters exposed to MS15 with concurrent subcutaneous injection of devazepide or vehicle. Litters were undisturbed after P10 and were weaned on P21. Subsets of adolescent males from each litter were tested in the EPMZ on P40-41, while others were tested for NSF on P50-52. As predicted, rats with a developmental history of MS15 displayed reduced anxiety-like behavior in the EPMZ and NSF tests. The anxiolytic effect of MS15 was preserved in vehicle-treated rats, but was reversed in devazepide-treated rats. These results support the view that endogenous CCK-1 receptor signaling in infants is a potential pathway through which maternal-pup interactions regulate the development and functional organization of emotional circuits that control anxiety-like behavior in the offspring.