These studies tested for a facilitatory interaction between noradrenergic and excitatory amino acid mechanisms controlling oxytocin (OT) release in the lactating rat. Lactating females were cannulated in the supraoptic nucleus of the hypothalamus (SON) or into the third ventricle and treated with the alpha 1-agonist phenylephrine (PHE) or the glutamate receptor agonist alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), either alone or together. Treatment with PHE increased plasma OT dose dependently after microinjection into the SON area; strong stimulation also occurred after third ventricle injection of the drug. AMPA caused dose-dependent increases in plasma OT after SON injection. Coinjection of an ineffective or submaximally effective dose of AMPA and a submaximally stimulating dose of PHE produced synergistic OT discharges. OT release in response to the combination of PHE plus AMPA could be abolished by pretreatment/cotreatment with either an alpha 1-adrenergic antagonist or an AMPA receptor antagonist. Moreover, the OT secretory response to the alpha 1-adrenergic agonist PHE alone was attenuated by blockade of AMPA receptors, whereas the OT secretory response to the glutamate agonist AMPA alone was attenuated by blockade of alpha 1-adrenergic receptors. These findings suggest an interaction between norepinephrine and glutamate that may involve pre- and/or postsynaptic mechanisms. As disruption of either noradrenergic or glutamatergic mechanisms is known to impair suckling-induced OT release, the cooperative action of transmitters active at alpha 1-adrenergic and AMPA receptors may be important for the milk ejection reflex.