Recent evidence suggests that the endogenous opioid peptides (EOPs) inhibit luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by suppression of hypothalamic gonadotropin-releasing hormone (GnRH) release, and that the feedback inhibition by EOPs is influenced by ovarian steroids. In the present studies, intact (INT) and ovariectomized (OVX) adult female rabbits were fitted with femoral vein catheters and mediobasal hypothalamic (MBH) push-pull perfusion (PPP) cannulae. One week after brain cannulation, does were subjected to 6 h of PPP and sequential blood sampling. In experiment I, INT (n = 6) and OVX (n = 5) does were infused intravenously with saline for 4 h followed by 2 h of infusion of the opiate antagonist naloxone (NAL; 10 µg/min/kg) while the MBH was simultaneously perfused with media. In experiment II, INT (n = 5) and OVX (n = 5) does were perfused with media for 4 h followed by 2 h of intrahypothalamic (IHP) NAL perfusion (0.2 µg/min). The GnRH in push-pull perfusates and LH and FSH in plasma samples collected at 10-min intervals were measured by specific radioimmunoassays. In INT does, neither intravenous infusion nor IHP perfusion of NAL altered pulsatile parameters of GnRH or LH release. In contrast, both intravenous and IHP NAL administration stimulated GnRH and LH release within 30–50 min in OVX does by marked increases in both GnRH and LH pulse amplitudes. Neither route of NAL administration affected FSH secretion in any of the treatment groups. We conclude that (1) EOPs are involved in the inhibition of hypothalamic GnRH secretion in OVX does; (2) the feedback inhibition by ovarian steroids on the hypothalamic-pituitary axis in the rabbit is sufficient to compromise the effects of EOPs, and (3) under these experimental conditions, the hypothalamic mechanisms which regulate the secretion of pituitary LH and FSH may be independ