A role for neuropeptide Y (NPY) in neurotransmission in rat iridial arterioles has been investigated. Reverse transcription-polymerase chain reaction analysis has demonstrated mRNA expression for both Y₁ and Y₂ receptors in the superior cervical ganglion and iris. The Y₁ agonist [Leu³¹,Pro³⁴]NPY caused a dose-dependent constriction of iris arterioles (50% effective concentration of 10⁸ M), but, at low concentrations (10⁹ and 10¹⁰ M), it failed to potentiate either submaximal responses to norepinephrine (10⁶ M) or submaximal, noradrenergic responses to nerve stimulation. In contrast, 10⁷ M [Leu³¹,Pro³⁴]NPY potentiated submaximal, noradrenergic responses to nerve stimulation (10 Hz, 1 s) and to a concentration of norepinephrine (10⁷ M) which produced only small contractions. The Y₁ antagonist 1229U91 blocked contractions induced by [Leu³¹,Pro³⁴]NPY. Stimulation of the nerves for longer periods (10 or 20 Hz; 5, 30, or 60 s) revealed a component of the response which was reduced by 1229U91. This component was not apparent after brief stimuli (10 Hz, 1 s), even when opposing receptor pathways were blocked. The Y₂ agonist N-acetyl-[Leu²⁸,Leu³¹]NPY^24-36 had little effect on arterioles preconstricted with either high potassium or an ₂-adrenoceptor agonist, or on nerve-mediated contractions. Results suggest that NPY, released from sympathetic nerves during long-duration, high-frequency stimulation, activates Y₁ receptors on iris arterioles to produce vasoconstriction and to potentiate responses to low concentrations of norepinephrine.