We used the patch-clamp technique to study the effect of H₂O₂ on the apical ROMK-like small conductance K (SK) channel in the CCD. Addition of H₂O₂ decreased the activity of the SK channels and the inhibitory effect of H₂O₂ was larger in the CCD from rats on a K-deficient (KD) diet than that on normal K or high K (HK) diet. However, application of H₂O₂ did not inhibit the SK channels in inside-out patches. We next explored the role of PTK in mediating the effect of H₂O₂ on SK channels. Application of H₂O₂ stimulated the activity of endogenous PTK in M-1 cells and increased tyrosine phosphorylation of ROMK in HEK293 cells transfected with GFP-ROMK1 and c-Src. However, blockade of PTK only attenuated but did not completely abolish the inhibitory effect of H₂O₂ on SK channels. We have also examined the role of P38 and ERK in mediating the effect of H₂O₂ on SK channels. Similar to blockade of PTK, suppression of P38 and ERK did not completely abolish the hydrogen peroxide induced inhibition of SK channels. However, combined use of ERK, P38 and PTK inhibitors completely abolished the effect of H₂O₂ on SK channels. Also, treatment of the CCDs with concanavalin A, an agent which has been shown to inhibit endocytosis, abolished the inhibitory effect of H₂O₂. We conclude that addition of H₂O₂ inhibited SK channels by stimulating PTK activity, P38 and ERK in the CCD and that H₂O₂ enhances the internalization of the SK channels.