The possible cooperation of neuropeptide Y (NPY) and alpha-1-adrenergic mechanisms in the release of oxytocin (OT) in conscious, nonsuckled lactating rats was examined following microinjections of NPY and its analogs and/or alpha-adrenergic drugs into the supraoptic nucleus (SON) or anterior paraventricular nucleus/anterior commissural nucleus (PVN/ACN). The alpha-1-adrenergic agonist phenylephrine dose dependently increased plasma OT after injection into the SON or the PVN/ACN, and this was prevented by treatment with the specific alpha-alpha-1-adrenergic receptor antagonist prazosin, but not by the alpha-2 antagonist rauwolscine. The alpha-2-adrenergic agonist clonidine did not increase plasma OT. Injection of NPY or of the related peptide YY (PYY) alone into the SON or the PVN/ACN also dose dependently increased plasma OT; this was also significantly attenuated by prazosin. Plasma OT responses to NPY and PYY differed significantly in dynamics and duration, which may be related to large differences found in the patterns of displaceable binding of [125I]NPY and [125I]PYY to hypothalamic membranes. The stimulatory action of NPY was mimicked by a preferential Y-1 receptor agonist, but not by an agonist of the Y-2 NPY receptor. Coinjection of NPY or PYY and phenylephrine into either the SON or the PVN/ACN area produced a greater than additive discharge of OT, especially in the SON. This increase was mostly due to a markedly enhanced initial release of OT, and was also completely eliminated by prazosin. The NPY-alpha-1 interaction in stimulating plasma OT was not mediated by direct binding of the peptides to alpha-1-adrenergic receptor sites. These results indicate that 1) the stimulatory noradrenergic influence on OT secretion in the lactating rat is mediated by the alpha-1 adrenergic receptor via an action in the PVN/ACN and SON; 2) NPY also stimulates OT secretion in the lactating rat through the Y-1 receptor subtype in the PVN/ACN and SON; 3) NPY markedly enhances the OT secretory responses to alpha-1 receptor stimulation, particularly in the SON. The cooperative stimulation by NE and NPY may be of physiological importance in mediating the episodic release of OT in response to suckling.