Chronic hyperprolactinemia in the rat is associated with suppression of plasma gonadotropin concentrations. The reduction of gonadotropins is considered to be due to either an inhibitory effect of prolactin (PRL) on pituitary gonadotropin secretion and/or suppression of hypothalamic gonadotropin-releasing hormone (GnRH) release. In contrast to the in vivo effects of PRL on hypothalamic GnRH release, it has recently been reported that endogenous hypothalamic PRL exerts a tonic stimulatory effect on GnRH release in vitro. The present study was undertaken to further evaluate the role of PRL on GnRH release. To accomplish this, we set up a static hypothalamic organ culture system which enabled us to evaluate immunoreactive GnRH (iGnRH) release by individually incubated longitudinally halved hypothalami. PRL at the concentrations of 100 and 1,000 nM (2,300 and 23,000 ng/ml) inhibited iGnRH release. This suppressive effect of PRL was apparently specific since growth hormone, a PRL-related molecule, did not have any significant effect on iGnRH release at the concentration of 1,000 nM (21,500 ng/ml) and a PRL antiserum completely suppressed the inhibitory effect of exogenously added PRL. On the other hand, this antiserum had no effect on basal iGnRH release suggesting that hypothalamic PRL does not regulate GnRH release. Based on the observation that PRL exerts a sexually dimorphic effect on plasma gonadotropin levels, we also evaluated the effects of testosterone (T), dihydrotestosterone (DHT), 17 beta-estradiol (E2), and progesterone (P) on PRL-inhibited iGnRH release in vitro. T, DHT and E2 did not have any detectable effect on PRL-suppressed iGnRH release whereas P, at the concentration of 0.1 nM (0.03 ng/ml), completely abolished the effect of PRL.(ABSTRACT TRUNCATED AT 250 WORDS)