A heterologous radioimmunoassay for canine prolactin was developed and used to study the role of catecholamines and indoleamines in the regulation of prolactin secretion in dogs. Female dogs were found to have a higher plasma prolactin concentration than male dogs. Thyrotropin-releasing hormone (TRH) increased plasma prolactin in male and female trained conscious dogs. L-Dopa blocked the increase produced by TRH, but this effect could be reversed by pretreatment with carbidopa, an inhibitor of the conversion of L-dopa to catecholamines outside the blood-brain barrier. The intravenous administration of the immediate precursor of serotonin, L-5-hydroxytryptophan (5HTP), caused an increase in plasma prolactin and growth hormone. The administration of the serotonin receptor blocker, metergoline, 18 and 3 h before 5HTP, significantly augmented the growth hormone response to 5HTP. In contrast, metergoline diminished the prolactin response to 5HTP. Pretreatment with carbidopa did not change the prolactin or growth hormone responses to 5HTP. These observations indicate that the dog is similar to other mammals in that females have greater plasma prolactin concentrations than males and TRH increases plasma prolactin. L-Dopa appears to act at a site outside the blood-brain barrier to prevent the prolactin response to TRH. The data suggest that 5HTP increases plasma prolactin via a serotonergic mechanism, whereas it increases plasma growth hormone via a different, nonserotonergic mechanism.