Soluble guanylyl cyclase (sGC), an enzyme involved in cGMP signal transduction, is activated by NO binding to the endogenous heme. The mechanism of deactivation is not known. In tissues, cGMP levels decrease within minutes, despite the fact that sGC is activated to levels above the phosphodiesterase activity. Simple dissociation of NO from the heme in sGC has been suggested as a possible deactivation mechanism; however, dissociation rates of NO from ferrous heme proteins are typically very slow. Since oxidants and reductants are known to affect sGC activity, we have tested the effect of a variety of redox-active agents on the activity of NO-activated sGC. All the redox-active compounds tested, covering a wide range of reduction potentials, selectively deactivated the NO-activated sGC while having little or no effect on the basal activity of the enzyme. Among the reagents studied in detail, deactivation of sGC by air occurred slowly, while deactivation by ferricyanide was faster and methylene blue was fastest. The mechanism of deactivation of sGC by dioxygen in the air is straightforward: the heme is oxidized to Fe(III)heme and nitrate is formed. This reaction is similar to that of dioxygen with NOHb and NOMb as occurs in cured meats. Methylene blue and ferricyanide deactivate sGC by a different, as yet undetermined, mechanism.