Serotonergic and histaminergic neuronal systems are both involved in mediation of the stress-induced release of thepituitary hormones prolactin (PRL) and ACTH. We investigated the possibility of an interaction between serotonin (5-HT) and histamine (HA) in regulation of PRL and ACTH secretion in conscious male rats. Animals were pretreated systemically with antagonists to 5-HT₁, 5-HT₂ or 5-HT3 receptors prior to intracerebroventricular (icv) administration of HA. The 5-HT₁₊₂ receptor antagonist methysergide prevented and the 5-HT₂ receptor antagonist LY 53857 attenuated the HA-induced PRL release while the 5-HT₃ receptor antagonist ondansetron had no effect on this response. None of the three 5-HT receptor antagonists affected the ACTH response to HA. Specific blockade of HA synthesis by α-fluoromethylhistidine or blockade of postsynaptic HA receptors byicv infusion of the H₁ receptor antagonist mepyramine or the H₂ receptor antagonist cimetidine inhibited the PRL response to 5-HT or to the 5-HT precursor 5-hydroxytryptophan (5-HTP) given in combination with the 5-HT reuptakeinhibitor fluoxetine (Fix). Blockade of the histaminergic system had no effect on the ACTH response to serotonergic stimulation. The H3 receptors are inhibitory HA receptors. Systemic pretreatment with the H₃ receptor agonist R(α)methylhistamine, or the H3 receptor antagonist thioperamide had no effect on the hormone response to activation of the serotonergicsystem by 5-HTP plus Flx. We conclude that the serotonergic and histaminergic neuronal systems interact in their stimulationof PRL secretion, but not in their stimulation of ACTH secretion. This interaction involves serotonergic 5-HT₁ and 5-HT₂ receptors and histaminergic H₁ and H₂ receptors. Furthermore, the previously observed inhibitory effect of the H₃ receptor agonist R(α)methylhistamine on stress-induced PRL and ACTH release seems not to be exerted by activation of presynaptic H₃ receptors located on serotonergic neurons but rather on histaminergic neurons.