The redox state of the heme of soluble guanylate cyclase (sGC) may regulate the sensitivity of vascular tissue to nitric oxide (NO). In this study, diphenyliodonium (DPI) is used as an inhibitor of flavoprotein oxidoreductases to examine their potential role in the expression of NO-elicited cGMP-associated arterial relaxation and sGC stimulation. The relaxation of endothelium-removed bovine coronary arteries (BCAs) precontracted with 30 mmol/L KCl to the NO donorS-nitroso-N-acetyl-penicillamine (SNAP) or to NO is markedly suppressed by 10 &mgr;mol/L DPI under an atmosphere of 21% O2(5% CO2). In contrast, DPI has minimal effects on the relaxation to SNAP under 95% N2(5% CO2). If BCAs are treated with DPI under 21% O2and then exposed to the hemoprotein reductant sodium dithionite (1 mmol/L) under N2, there is a partial reversal of the inhibitory effects of DPI compared with BCAs that were not treated with dithionite. DPI did not inhibit relaxation elicited by 8-bromo-cGMP or forskolin. Increases in tissue cGMP levels stimulated by SNAP were eliminated by pretreatment of BCAs with DPI under 21% O2but not under N2. Activation of sGC by SNAP in BCA homogenate was also eliminated when vessels were pretreated with 10 &mgr;mol/L DPI under 21% O2, but DPI did not have an inhibitory effect when directly added to the assay of sGC activity. These observations are consistent with a flavoprotein-dependent oxidoreductase functioning to prevent the expression of a novel O2-dependent process from oxidizing the heme on sGC and inhibiting NO-elicited cGMP-mediated BCA relaxation.