Progesterone (P) stimulates prolactin secretion through an unknown neural mechanism in estrogen (E)-primed female monkeys. Serotonin also stimulates prolactin secretion and this laboratory demonstrated that E induces nuclear progestin receptors (PR) in serotonin neurons. Thus, PR in serotonin neurons could transduce the action of P on prolactin secretion. Studies were performed to determine (1) whether blocking nuclear PR would block P-induced prolactin secretion and conversely; (2) whether increasing serotonin concentrations in the synapse would augment P-induced prolactin secretion. In both studies, female monkeys were spayed, adapted to a vest and tether remote sampling system and catheterized prior to experiments. Monkeys received 2 E-filled silastic implants (3.0 cm) 1-3 weeks prior to study. P (20 mg) in corn oil was injected (s.c.) to transiently increase prolactin secretion. In both studies, each monkey served as its own control. To block nuclear PR and not membrane PR, RU 486 (2 mg/kg, i.m.) or ethanol (control) was administered with the P injection. Relative to the P injection, blood samples were taken twice daily from -30 to +24 h, then every 4 h from +36 to +48 h and once at +65 h. To increase serotonin in the synapse, the serotonin reuptake inhibitor, fluoxetine (5 mg/day, i.v.), was infused for 4 weeks. P was injected during the week of vehicle infusion and during the last week of fluoxetine infusion. Blood samples were obtained twice daily prior to and following P treatment. Prolactin, E, P and RU 486 concentrations were determined by RIA. RU 486 completely blocked the P-induced prolactin surge (n = 3). In addition, fluoxetine significantly increased prolactin secretion during the P-induced prolactin peak compared to equal time points during saline infusion (n = 5). These data indicate that P induces prolactin via a genomic mechanism and not through a membrane action. The data also support a pivotal role for serotonin in the neural regulation of P-induced prolactin secretion.