We previously demonstrated that nitric oxide (NO) stimulates the basolateral small-conductance K⁺channel (SK) via a cGMP-dependent pathway [M. Lu and W. H. Wang.Am. J. Physiol. 270 (Cell Physiol. 39): C1336–C1342, 1996]. Because NO at high concentration has been shown to react with superoxide () to form peroxynitrite (OONO⁻) [W. A. Pryor and G. L. Squadrito. Am. J. Physiol. 268 (Lung Cell. Mol. Physiol. 12): L699–L722, 1995 and M. S. Wolin.Microcirculation 3: 1–17, 1996], we extended our study to examine, using patch-clamp technique, the effect of high concentrations of NO on SK in cortical collecting duct (CCD) of rat kidney. Addition of NO donors [100–200 μMS-nitroso-N-acetyl-penicillamine (SNAP) or sodium nitroprusside (SNP)] reduced channel activity, defined as the product of channel number and open probability, to 15 and 25% of the control value, respectively. The inhibitory effect of NO was completely abolished in the presence of 10 mM Tiron, an intracellular scavenger of . NO donors, 10 μM SNAP or SNP, which stimulate channel activity under control conditions, can also inhibit SK in the presence of an donor, pyrogallol, or in the presence of an inhibitor of superoxide dismutase, diethyldithiocarbamic acid. The inhibitory effect of NO is still observed in the presence of exogenous cGMP, suggesting that the NO-induced inhibition is not the result of decreased cGMP production. We conclude that the inhibitory effect of NO on channel activity results from an interaction between NO and .