S-Nitrosoglutathione reductase (GSNOR) is an alcohol dehydrogenase involved in the regulation of S-nitrosothiols (SNOs) in vivo. Knock-out studies in mice have shown that GSNOR regulates the smooth muscle tone in airways and the function of beta-adrenergic receptors in lungs and heart. GSNOR has emerged as a target for the development of therapeutic approaches for treating lung and cardiovascular diseases. We report three compounds that exclude GSNOR substrate, S-nitrosoglutathione (GSNO) from its binding site in GSNOR and cause an accumulation of SNOs inside the cells. The new inhibitors selectively inhibit GSNOR among the alcohol dehydrogenases. Using the inhibitors, we demonstrate that GSNOR limits nitric oxide-mediated suppression of NF-kappaB and activation of soluble guanylyl cyclase. Our findings reveal GSNOR inhibitors to be novel tools for regulating nitric oxide bioactivity and assessing the role of SNOs in vivo.