The unilateral microinjection of the cholinergic agonist carbachol (CCh) or the biologically active peptide neuropeptide Y (NPY) into the posterior hypothalamic nucleus (PHN) of conscious, freely-moving rats evokes a pressor response which is mediated primarily by sympathoexcitation. Based on the ability of these substances to induce a pressor response via sympathoexcitation and the ability of NPY to modify responses evoked by several other neurotransmitters, the present study was undertaken to determine if NPY can alter the pressor response evoked by subsequent administration of CCh into the PHN. Microinjection of CCh into the PHN in six doses ranging from 0.4 to 11 nmol induced a dose-dependent increase in mean arterial pressure (MAP) and area under the curve of the change in MAP. Administration of 0.23 nmol of NPY into the PHN induced a small but significant increase in MAP when compared to the change observed after the microinjection of 0.9% saline. This NPY-induced increase was blocked by pretreatment with 2.3 nmol of the Y receptor antagonist PYX-2. Administration of 0.23 nmol of NPY 60 min prior to CCh induced a parallel shift to the left of the dose-response curves for CCh resulting in an increase in relative potency for CCh of 6.4 to 6.9 times. This NPY-mediated enhancement was prevented by pretreatment with PYX-2 which alone did not affect the CCh-induced pressor response. These results show that NPY enhances the pressor response evoked by CCh administration into the PHN of the conscious rat and that this enhancement is mediated by stimulation of a Y receptor.