Despite evidence for an important role for endothelium-derived relaxing factor (EDRF) in transitional circulation, previous in vitro studies of newborn pulmonary arteries have demonstrated diminished EDRF activity when compared with arteries from older animals. We studied pulmonary arteries and veins isolated from early newborn and juvenile sheep using standard tissue bath techniques. Incubation of vessels with the nitric oxide synthase inhibitor N omega-nitro-L-arginine (L-NNA) constricted veins but not arteries from both age groups. Further studies using preconstricted vessels revealed that arteries relaxed to acetylcholine (ACh), with significantly greater responses observed in juvenile arteries. Veins from both age groups contracted to ACh. Pretreatment with prostaglandin inhibitors (indomethacin and SQ 29,548) diminished ACh relaxations in pulmonary arteries from both age groups, greatly enhanced relaxations to ACh in newborn pulmonary veins, and depressed contractions in juvenile pulmonary veins. Removal of endothelium mechanically or functionally with prostaglandin inhibitors and L-NNA eliminated relaxations to ACh in pulmonary arteries from both age groups and resulted in contractions in veins. We conclude that isolated pulmonary veins from newborn sheep exhibit both baseline and stimulated release of EDRF, and we speculate that these venous responses may be important in the transitional pulmonary circulation.