In a variety of systemic blood vessels, protein kinase G (PKG) plays a critical role in mediating relaxation induced by agents that elevate cGMP, such as nitric oxide. The role of PKG in nitric oxide- and cGMP-induced relaxation is less certain in the pulmonary circulation. In the present study, we examined the effects of inhibitors of PKG on the responses of isolated fourth-generation pulmonary veins of newborn lambs (10 +/- 1 days of age) to nitric oxide and cGMP. In vessels preconstricted with endothelin-1, nitric oxide and 8-bromo-cGMP (a cell-membrane-permeable cGMP analog) induced concentration-dependent relaxation. The relaxation was significantly attenuated by beta-phenyl-1, N(2)-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothionate (Rp-8-Br-PET-cGMPS; a PKG inhibitor) and N-[2-(methylamino)ethyl]5-isoquinolinesulfonamide [H-8; an inhibitor of PKG and protein kinase A (PKA)] but was not affected by KT-5720 (a PKA inhibitor). Biochemical study showed that PKG activity in newborn ovine pulmonary veins was inhibited by 8-Br-PET-cGMPS and H-8 but not by KT-5720. PKA activity was not affected by 8-Br-PET-cGMPS but was inhibited by H-8 and KT-5720. These results suggest that PKG is involved in relaxation of pulmonary veins of newborn lambs induced by nitric oxide and cGMP.