In order to ascertain if prolactin (PRL) secretion stimulated by serotonin (5-HT) was mediated by the dopaminergic system, both dopamine (DA) and 5-HT were infused into the third ventricle of anesthetized laying turkeys in conjunction with the D1 DA receptor antagonist R(+)-SCH-23390 HCl or the 5-HT receptor antagonist Mianserin HCl. When infused alone at the rate of 10 nmol/min, both DA and 5-HT increased circulating PRL levels significantly (P < 0. 05) after 20 min (111.0 +/- 19.0 to 334.0 +/- 115.1 ng/ml and 93.3 +/- 13.2 to 173.8 +/- 33.3 ng/ml, respectively). When infused in conjunction with the D1 DA antagonist, the stimulatory effect of both DA and 5-HT on PRL release was significantly suppressed. The 5-HT antagonist completely blocked 5-HT-stimulated PRL release when infused at the rate of 10 nmol/min. When the 5-HT antagonist was co-infused with DA, PRL levels significantly increased (33.5 +/- 3.3 to 112.2 +/- 19.7 ng/ml) after 30 min, an increase that did not differ significantly from that caused by DA alone (63.2 +/- 16.4 to 144.5 +/- 44.1 ng/ml). These data suggest that 5-HT stimulation of PRL secretion requires an intact dopaminergic system and that 5-HT synapses are located further upstream than DA synapses along the PRL-releasing pathway within the avian hypothalamus.