Revised Manuscript Received November 12, 1992 abstract: A labile inorganic free radical, nitric oxide (’NO), is produced by nitric oxide synthase from the substrate L-arginine in various cells and tissues. It acts as an endothelium-derived relaxing factor (EDRF) or as a neurotransmitter in vivo. We investigated the reactivity of stable radical compounds, imidazolineoxyl A-oxides such as 2-phenyl-4, 4, 5, 5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), carboxy-PTIO, and carboxymethoxy-PTIO against'NO/EDRF in both chemical and biological systems. By using electron spin resonance (ESR) spectroscopy, imidazolineoxyl TV-oxides were found to react with’NO in a stoichiometric manner (PTIO/’NO= 1.0) in a neutral solution (sodium phosphate buffer, pH 7.4) with rate constants of~ 104 M_1 s" 1, resulting in the generation of N02_/N03_ and imidazolineoxyls such as 2-phenyl-4, 4, 5, 5-tetramethylimidazoline-1-oxyl (PTI), carboxy-PTI, or carboxymethoxy-PTI. Furthermore, the effects of imidazolineoxyl iV-oxides on acetylcholine-or ATP-induced relaxation of the smooth muscle of rabbit aorta were tested. The vasorelaxations were inhibited by all three imidazolineoxyl 7V-oxides markedly. The inhibitory effects of carboxy-PTIO was almost 2-fold stronger than those of’NO synthesis inhibitors, jV “-nitro-L-arginine and 7V “-monomethyl-L-arginine. Generation of EDRF/'NO was identified by reacting the PTIO in aortic strips and quantitating the reaction product with ESR spectroscopy. Thus, it was clarified that imidazolineoxyl 7V-oxide antagonize EDRF/’NO via a unique radical-radical reaction with’NO.