Since prolactin (PRL) can increase electrically stimulated oxytocin (OT) release by a direct action on the neurohypophysis, experiments were done to test the effect of PRL on OT mRNA content of explants of the hypothalamo-neurohypophyseal system (HNS) obtained from Day 10 lactating Sprague-Dawley rats, to determine if PRL could alter OT mRNA by a direct effect on the HNS. The effect of PRL was evaluated alone and in conjunction with bicuculline, an antagonist at gamma amino butyric acid receptors, in order to provide a concomitant stimulus for OT release. Neither PRL nor bicuculline alone altered OT or VP release. However, the simultaneous administration of bicuculline and PRL caused a statistically significant increase in the release of OT and in OT mRNA content of the explants (P < 0.05). On the other hand, PRL did not cause vasopressin (VP) release nor an increase in VP mRNA in these explants even in the presence of bicuculline, thus ruling out a direct effect of PRL on the HNS VP neurons. In conclusion, during lactation, PRL, in combination with other stimuli can increase OT mRNA content in the hypothalamus by a direct action on the hypothalamo-neurohypophyseal system. This effect may be secondary to the release of OT. This effect is specific to OT, because PRL did not alter either the release of VP or VP mRNA content.