The intrahypothalamic site(s) of endogenous opioid regulation of GnRH secretion remains to be resolved. Accordingly, we used an in vitro acute incubation system to evaluate GnRH, dopamine (DA), and norepinephrine (NE) release from adult male rat median eminences (MEs) in response to the opiate receptor agonist morphine (MOR) and the opiate receptor antagonist naloxone (NAL). MOR (2 mM) stimulated basal and K+-induced GnRH release from isolated MEs, but 0.25, 5, or 100 microM MOR was without significant effect. NAL (1 mg/ml; 2.8 mM) increased basal GnRH release, but 0.01 mg NAL/ml suppressed basal GnRH release, and neither 0.001 nor 0.1 mg NAL/ml had an appreciable effect. NAL did not significantly alter K+-induced GnRH release. In a separate experiment, 1 mg NAL/ml stimulated but 0.01 mg NAL/ml inhibited basal release of DA and NE from the ME. NAL (1 ng/ml) also decreased K+-induced DA and NE release. The rates of basal and K+-induced DA and NE release were highly correlated with GnRH release during corresponding 0, 0.01, and 1.0 mg/ml NAL treatments in the preceding experiment (r = 0.98 and 0.93, respectively). Thus, 2 mM MOR stimulated but different NAL dosages either stimulated or inhibited GnRH release from isolated MEs, suggesting complex opioid regulation at the level of the GnRH neurosecretory terminals. The precise correlation between GnRH and DA/NE release suggests that the catecholamine terminals close to both GnRH- and endorphin-containing terminals in the ME may mediate this opioid regulation.