The role of membrane-bound protein serine/threonine phosphatases (PP) in modulating the renal ATP-sensitive K+ (K(ATP)) channel was examined using the patch-clamp technique in principal cells of rat cortical collecting duct. In the absence of ATP, channel activity rapidly (11.2 s) declines (channel 'rundown') upon excision of the membrane patches into control bath solutions (1 mM Mg2+, Ca2+ free). Both orthovanadate (5 mM), a broad-spectrum inhibitor of phosphatases except for Ca2+-dependent PP (PP-2B), and okadaic acid (OA, 1 μM), a potent inhibitor of PP types 1 and 2A (PP-1 and PP-2A), significantly slowed channel rundown. Removal of Mg2+ from the bath also slowed the rundown process. Incubation of cells with OA in the absence of Mg2+ or with orthovanadate in ATP- free solution maintained channel activity at levels of ~ 70% of control values for 3 min after membrane excision. In contrast, Ca2+ (0.1 mM) and calmodulin (1 μM) in the presence of 1 mM Mg2+, a condition in which PP-2B is stimulated, had no significant effect on the channel activity that persisted in the presence of OA and orthovanadate. Application of exogenous PP-2A (1 U/ml) to the cytosolic side of membrane in inside-out patches significantly inhibited channel activity to 35.0%, of control, but the inhibitory effects of PP-1 (1 U/ml) and PP-2B (20 μg/ml) were minor. These results suggest that rundown of the renal K(ATP) channel after membrane excision results mainly from dephosphorylation of the channel or an associated protein by membrane-bound phosphatases. Our data suggest that at least two types of phosphatases. OA-sensitive PP-2A and a Mg2+-dependent phosphatase, possibly PP-2C, are involved in this dephosphorylation process.