—G_s protein–coupled β-adrenoceptors rapidly desensitize on exposure to agonists in reconstituted membrane preparations, whereas rapid tachyphylaxis to β-adrenoceptor–mediated vasodilation does not readily occur in vivo. This study examined the possibility that endothelium-derived nitrosyl factors prevent the rapid desensitization of β-adrenoceptors in the vascular smooth muscle of resistance arteries in pentobarbital-anesthetized rats. The fall in mean arterial blood pressure and in hindquarter vascular resistance produced by the β-adrenoceptor agonist isoproterenol (ISO, 0.1 to 10 μg/kg IV) was slightly but significantly smaller in rats treated with the NO synthase inhibitor N^G-nitro-l-arginine methyl ester (L-NAME, 100 μmol/kg IV) than in saline-treated rats. The ISO-induced fall in mesenteric resistance was similar in L-NAME–treated and in saline-treated rats. The fall in hindquarter vascular resistance and in mesenteric resistance produced by ISO (8×10 μg/kg IV) was subject to tachyphylaxis on repeated injection in rats treated with L-NAME (100 μmol/kg IV) but not in rats treated with saline. Injections of l-S-nitrosocysteine (1200 nmol/kg IV), a lipophobic S-nitrosothiol, before each injection of ISO (10 μg/kg IV) prevented tachyphylaxis to ISO in L-NAME–treated rats. The vasodilator effects of ISO (0.1 to 10 μg/kg IV) in L-NAME–treated rats that received 8 injections of ISO (10 μg/kg IV) were markedly smaller than in L-NAME–treated rats that received 8 injections of saline. These results indicate that (1) the vasodilator actions of ISO in pentobarbital-anesthetized rats only minimally involve the release of endothelium-derived nitrosyl factors, (2) the effects of ISO are subject to development of tachyphylaxis in L-NAME–treated rats, and (3) tachyphylaxis to ISO is prevented by l-S-nitrosocysteine. These findings suggest that endothelium-derived nitrosyl factors may prevent desensitization of β-adrenoceptors in vivo.