Previous studies have demonstrated that an increase in the activity of protein-tyrosine kinase (PTK) is involved in the down-regulation of the activity of apical small conductance K+ (SK) channels in the cortical collecting duct (CCD) from rats on a K+-deficient diet (1). We used the patch clamp technique to investigate the role of protein-tyrosine phosphatase (PTP) in the regulation of the activity of SK channels in the CCD from rats on a high K+ diet. Western blot analysis indicated that PTP-1D is expressed in the renal cortex. Application of 1 μM phenylarsine oxide (PAO) or 1 mM benzylphosphonic acid, agents that inhibit PTP, reversibly reduced channel activity by 95%. Pretreatment of CCDs with PAO for 30 min decreased the mean NP reversibly from control value 3.20 to 0.40. Addition of 1 μM herbimycin A, an inhibitor of PTK, had no significant effect on channel activity in the CCDs from rats on a high K+ diet. However, herbimycin A abolished the inhibitory effect of PAO; indicating that the effect of PAO is the result of interaction between PTK and PTP. Addition of brefeldin A, an agent that blocks protein trafficking from Golgi complex to the membrane, had no effect on channel activity. Moreover, application of colchicine, a microtubule inhibitor, or paclitaxel, a microtubule stabilizer, had no effect on channel activity. In contrast, PAO still reduced channel activity in the presence of brefeldin A, colchicine, or paclitaxel. Furthermore, the effect of PAO on channel activity was absent when the tubules were bathed in 16% sucrose- containing bath solution or treated with concanavalin A. We conclude that PTP is involved in the regulation of the activity of SK channels and that inhibition of PTP may facilitate the internalization of the SK channels.