Corticotropin-releasing hormone (CRH) has been shown capable of inhibiting hypothalamic gonadotropin-releasing hormone (GnRH) release through activation of an endogenous opioid peptide (EOP)-dependent mechanism. Recently, we have shown that prolactin (PRL) stimulates CRH release and inhibits GnRH release by hypothalami explanted from male rats. Thus, the present study was undertaken to investigate whether the inhibitory effect of PRL on GnRH release in vitro is mediated by CRH and/or EOP. To this aim, the release of GnRH in response to PRL was evaluated in presence of CRH<sub>9–41</sub> α-helical (CRH<sub>9–41</sub>), a CRH receptor antagonist, and/or naloxone (NAL), a nonselective opioid receptor antagonist, using a static hypothalamic organ culture system which enabled us to evaluate immunoreactive GnRH (iGnRH) release from individually incubated longitudinally halved hypothalamic. As previously shown, PRL at the concentration of 100nmol/l inhibited basal iGnRH release by about 35%. CRH<Sub>9–41</Sub> or NAL overcame the inhibitory effect of PRL on iGnRH release in a concentration-dependent fashion. The simultaneous co-incubation with both antagonists was not more effective than each single antagonist. CRH<sub>9–41</sub> did not have any effect on basal iGnRH release whereas NAL, as previously reported, increased it. In addition, PRL at the concentration of 100 nmol/l stimulated basal hypothalamic β-endorphin (β-EP) release. In conclusion, these data show that antagonism to CRH receptors counteracts the suppressive effects of PRL upon GnRH release and that PRL is able to stimulate hypothalamic β-EP release in vitro.