Beta-adrenergic agonists and prostaglandin E2 (PGE2) play an important role in perinatal pulmonary circulation. We have determined the effect of antenatal glucocorticoid treatment on isoprenaline- and PGE2-mediated relaxation of pulmonary arteries of newborn preterm lambs. Ovine fetuses (121 days of gestation; term = 150 days) received a single intramuscular dose of betamethasone (0.5 mg/kg) or saline. Fifteen hours after the injection, the lambs were delivered, ventilated for 3 h, and sacrificed. The fourth-generation pulmonary arteries were dissected and cut into rings for study. In endothelin-1-preconstricted vessels, isoprenaline, PGE2, and forskolin (an activator of adenylyl cyclase) induced greater relaxations of pulmonary arteries of betamethasone-treated lambs than those of controls. 8-Bromo-cyclic adenosine monophosphate, a cell membrane permeable analogue of cyclic adenosine monophosphate, caused similar relaxation of all vessels. When stimulated with isoprenaline and PGE2, the adenylyl cyclase activity of crude membrane preparations of pulmonary arteries treated with betamethasone was greater than that of controls. These results show that single-dose antenatal betamethasone treatment enhances relaxation of pulmonary arteries of preterm lambs induced by isoprenaline and PGE2 and that an enhanced adenylyl cyclase activity contributes to the effect of betamethasone on pulmonary arteries of preterm lambs.