Continuous exposure to nitrovasodilators and nitric oxide induces tolerance to their vasodilator effects in vascular smooth muscle. This study was done to determine the role of cyclic GMP-dependent protein kinase (PKG) in the development of tolerance to nitric oxide. Isolated fourth generation pulmonary veins of newborn lambs were studied. Incubation of veins for 20 h with DETA NONOate (DETA NO; a stable nitric oxide donor) significantly reduced their relaxation response to the nitric oxide donor and to 8-Br-PET-cGMP (a cell permeable cGMP analog). Incubation with DETA NO significantly reduced PKG activity, protein and mRNA levels in the vessels. These effects were prevented by ODQ (an inhibitor of soluble guanylyl cyclase) and Rp-8-Br-PET-cGMPS (an inhibitor of PKG). A decrease in PKG protein and mRNA levels was also observed following continuous exposure to cGMP analogs. The PKG inhibitor abrogated these effects. The decrease in cGMP-mediated relaxation and in PKG activity caused by continuous exposure to DETA NO was not affected by KT-5720, an inhibitor of cAMP-dependent protein kinase (PKA). Prolonged exposure to 8-Br-cAMP (a cell permeable cAMP analog) did not affect PKG protein level in the veins. These results suggest that continuous exposure to nitric oxide or cGMP down-regulates PKG by a PKG-dependent mechanism. Such a negative feedback mechanism may contribute to the development of tolerance to nitric oxide in pulmonary veins of newborn lambs.