Dephosphorylation of the CFTR Cl- channel is known to be induced by both okadaic-acid- (OA-) sensitive and -insensitive protein phosphatases (PPs). In the present study, the effects of cytosolic free Mg2+ on the cardiac CFTR Cl- current were examined in relation to the latter PP activity in guinea pig ventricular myocytes. Even when maintaining intracellular Mg-ATP at millimolar concentrations under whole-cell patch-clamp mode, cAMP-activated Cl- conductance was reversibly suppressed by cytosolic free Mg2+, with an IC50 of around 2.5 mmol/l. In contrast, changes in the cytosolic concentration of free Mg2+ ([Mg2+](i)) had no effect on genistein-activated CFTR Cl- currents. The Mg2+ effect on cAMP-activated CFTR Cl- conductance was completely reversed by application of anthracene-9-carboxylic acid (9-AC), which was previously shown to inhibit an OA-insensitive PP in cardiac myocytes. A 9-AC-sensitive fraction of endogenous PP activity in the extract of guinea pig ventricle was found to be activated by free Mg2+ at millimolar concentrations but to be inactive at micromolar concentrations. The intracellular application of OA failed to activate basal Cl- conductance at millimolar [Mg2+](i). In the presence of OA, however, basal Cl- conductance became activated either by reducing [Mg2+](i) to micromolar concentrations or by applying 9-AC. Thus, we conclude that a Mg2+-dependent PP sensitive to 9-AC plays a key role in the cAMP-mediated regulation of cardiac CFTR Cl- channel at physiological [Mg2+](i) under both basal and cAMP-activated conditions. Also, it appears that the genistein-activated conformation of the cardiac CFTR channel is not sensitive to the Mg2+-dependent PP.