Pulsatile secretion of GnRH-1 regulates gonadotropin release from anterior pituitary and thus is essential for reproduction. The present study focused on the role of cholecystokinin (CCK) in the GnRH-1 system. CCK is a neuropeptide abundantly expressed in the brain, which is implicated in activation of female reproductive behaviors and release of anterior pituitary hormones. Using dual-label immunocytochemistry coupled to confocal analysis, GnRH-1 neurons in adult mouse brain were found to express CCK-1 receptors (CCK-1R), and CCK fibers were detected contacting GnRH-1 axons. To address the function of CCK on GnRH-1 neurons, calcium imaging was used to monitor patterns of activity of GnRH-1 neurons maintained in an in vitro system known to retain many characteristics of GnRH-1 cells in vivo. Endogenous receptors for CCK (CCK-1R and CCK-2R) were blocked with selective antagonists. Results indicate that CCK-1R but not CCK-2R antagonist treatment increased the number of calcium peaks/GnRH-1 cell, mean peak amplitude, and percentage of GnRH-1 cells displaying high activity. The increased activity in GnRH-1 neurons observed after application of CCK-1R antagonist was blocked by coincubation with exogenous CCK. This study provides evidence that CCK acts directly on GnRH-1 neurons to attenuate GnRH-1 neuronal activity via CCK-1R activation.