Activity of an inwardly rectifying K⁺ channel in cultured human proximal tubule cells (RPTECs) is stimulated and inhibited by nitric oxide (NO) at low and high concentrations, respectively. In this study, we investigated the effects of interferon- (IFN-), one of cytokines which affect the expression of inducible NO synthase (iNOS), on intracellular NO and channel activity of RPTECs, using RT-PCR, NO imaging and the cell-attached mode of the patch-clamp technique. Prolonged incubation (24 h) of cells with IFN- (20 ng/ml) enhanced iNOS mRNA expression and NO production. In these cells, a NOS inhibitor, N-nitro-L-arginine methyl ester (L-NAME; 100 µM) elevated channel activity, suggesting that NO production was so high as to suppress the channel. This indicated that IFN- would chronically suppress channel activity by enhancing NO production. Acute effects of IFN- was also examined in control cells. Simple addition of IFN- (20 ng/ml) to the bath acutely stimulated channel activity, which was abolished by inhibitors of IFN- receptor-associated janus activated kinase (P6; 1 µM and AG490; 10 µM). However, L-NAME did not block the acute effect of IFN-. Indeed, IFN- did not acutely affect NO production. Moreover, the acute effect was not blocked by inhibition of protein kinases A, G (PKA, PKG), and phosphatidylinositol 3-kinase (PI3K). We conclude that IFN- exerted a delayed suppressive effect on K⁺ channel activity by enhancing iNOS expression and an acute stimulatory effect which was independent of either NO pathways or phosphorylation processes mediated by PKA, PKG, and PI3K in RPTECs.