An inwardly rectifying, ATP-regulated K+ channel with a distinctive molecular architecture, ROMK1, was recently cloned from rat kidney. Using patch clamp techniques, we have investigated the regulation of ROMK1 with particular emphasis on phosphorylation/dephosphorylation processes. Spontaneous channel rundown occurred after excision of membrane patches into ATP-free bath solutions in the presence of Mg2+. Channel rundown was almost completely abolished after excision of patches into either Mg2+-free bathing solutions or after preincubation with the broad-spectrum phosphatase inhibitor, orthovanadate, in the presence of Mg2+. MgATP preincubation also inhibited channel rundown in a dose-dependent manner. In addition, the effect of the specific phosphatase inhibitors okadaic acid (1 μM) and calyculin A (1 μM) was also investigated. The presence of either okadaic acid or calyculin A failed to inhibit channel rundown. Taken together, these data suggest that rundown of ROMK1 involves a Mg2+-dependent dephosphorylation process. Channel activity was also partially restored after the addition of MgATP to the bath solution. Addition of exogenous cAMP-dependent protein kinase A (PKA) catalytic subunit led to a further increase in channel open probability. Addition of Na2ATP, in the absence of Mg2+, was ineffective, suggesting that restoration of channel activity is a Mg2+-dependent process. Addition of the specific PKA inhibitor, PKI, to the bath solution led to a partial, reversible inhibition in channel activity. Thus, PKA- dependent phosphorylation processes are involved in the modulation of channel activity. This observation is consistent with the presence of potential PKA phosphorylation sites on ROMK1.