The inwardly rectifying K+ channel with an inward conductance of about 90 pS in the surface membrane of cultured opossum kidney proximal tubule (OKP) cell is activated by cyclic AMP-dependent protein kinase (PKA). In this study, we further examined the involvement of the guanosine 3',5'-cyclic monophosphate (cGMP)-dependent process in modulation of this K+ channel by using the patch-clamp technique. In cell-attached patches, channel activity was increased by the application of either N2, 2'-O-dibutyrylguanosine 3',5'-cyclic monophosphate (DBcGMP, 100 microM) or 8-bromoguanosine 3',5'-cyclic monophosphate (8BrcGMP, 100 microM), and it was inhibited by KT5823 (10 microM), a membrane-permeable specific inhibitor of cGMP-dependent protein kinase (PKG). The effect of DBcGMP on channel activity was abolished by the pretreatment of cells with KT5823 (10 microM), but it was observed in the presence of KT5720 (200 nM), a specific inhibitor of PKA. Furthermore, atrial natriuretic peptide (ANP, 10 nM) increased channel activity, which was also prevented by the application of KT5823 (10 microM). In inside-out patches, ATP (3 mM) was required to maintain channel activity, which was inhibited by KT5823 (10 microM), but it was not increased by cGMP (100 microM) alone. The channel activity was increased by the coapplication of PKG (500 U/ml) and cGMP (100 microM). These results suggest that cGMP activates the inwardly rectifying K+ channel in OKP cells through PKG-mediated phosphorylation processes independent of PKA-mediated processes, and that ANP is an agonist which stimulates PKG-mediated processes in the proximal tubule cell. Furthermore, it is suggested that the ATP-dependent channel activity in inside-out patches is maintained at least in part by PKG, which is the membrane-bound catalytic domain.