We investigated the effects of nitric oxide (NO) on activity of the inwardly rectifying K⁺ channel in cultured human proximal tubule cells, using the cell-attached mode of the patch-clamp technique. An inhibitor of NO synthases, N-nitro-L-arginine methyl ester (L-NAME; 100 µM), reduced channel activity, which was restored by an NO donor, sodium nitroprusside (SNP; 10 µM) or 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP; 100 µM). However, SNP failed to activate the channel in the presence of an inhibitor of soluble guanylate cyclase, 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (10 µM). Similarly, the SNP effect was abolished by a PKG-specific inhibitor, KT-5823 (1 µM), but not by a PKA-specific inhibitor, KT-5720 (500 nM). Another NO donor, S-Nitroso-N-acetyl-D,L-penicillamine (10 µM), mimicked the SNP-induced channel activation. In contrast to the stimulatory effect of SNP at a low dose (10 µM), a higher dose of SNP (1 mM) reduced channel activity, which was not restored by 8-BrcGMP. Recordings of membrane potential with the slow whole-cell configuration demonstrated that L-NAME (100 µM) and the high dose of SNP (1 mM) depolarized the cell by 10.1 ± 2.6 mV and 9.2 ± 1.0 mV, respectively, whereas the low dose of SNP (10 µM) hyperpolarized it by 7.1 ± 0.7 mV. These results suggested that the endogenous NO would contribute to the maintenance of basal activity of this K⁺ channel and hence the potential formation via a cGMP/PKG-dependent mechanism, while a high dose of NO impaired channel activity independent of cGMP/PKG-mediated processes.