Endogenous nitric oxide (NO) donor compounds (S-nitrosothiols) contribute to low vascular tone by both cGMP-dependent and -independent pathways. We have reported that S-nitrosoglutathione (GSNO) inhibits 5-HT-mediated pulmonary vasoconstriction via a cGMP-independent mechanism likely involving S-nitrosylation of its G-protein coupled receptor (GPCR) system. Because catecholamines, like 5-HT, constrict lung vessels via a GPCR coupled to Gq, we hypothesized that S-nitrosothiols modify the ₁-adrenergic GPCR system to inhibit pulmonary vasoconstriction by receptor agonists, e.g. phenylephrine (PE). Rat pulmonary artery (PA) rings were pretreated for 30 minutes with and without an S-nitrosothiol, either GSNO or S-nitrosocysteine (CSNO), and constricted with sequential concentrations of PE (10^-8-10^-6 M). Effective cGMP-dependence was tested in rings pre-treated with soluble guanylate cyclase inhibitors (either ODQ or LY 83583), or G kinase inhibitor (KT 5823), and a thiol reductant (dithiothreitol, DTT) was used to test reversibility of S-nitrosylation. Both S-nitrosothiols attenuated the PE dose-response. The GSNO effect was not prevented by LY 83583, ODQ or KT 5823, indicating cGMP-independence. GSNO inhibition was reversed by DTT, consistent with S-nitrosylation or other GSNO-mediated cysteine modifications. In CSNO-treated lung protein, the ₁-adrenergic receptor was shown to undergo S-nitrosylation in vitro using a biotin switch assay. Studies of ₁-adrenergic receptor subtype expression and receptor density by saturation binding with [¹²⁵I] HEAT showed that GSNO decreased ₁-adrenergic receptor density but did not alter affinity for antagonist or agonist. These data demonstrate a novel cGMP-independent mechanism of reversible ₁-adrenergic receptor inhibition by S-nitrosothiols.