Prostacyclin is a pulmonary vasodilator and is produced by prostacyclin synthase and stimulates adenylate cyclase (AC) via the prostacyclin receptor (IP) to produce cAMP. Forskolin is a direct stimulant of AC. Phosphodiesterase 3 hydrolyzes cAMP and is inhibited by milrinone. OBJECTIVE:: To characterize the prostacyclin-AC-cAMP pathway in the ovine ductal ligation model of persistent pulmonary hypertension of the newborn (PPHN). SETTING:: University-based laboratory animal facility. SUBJECTS:: Lambs delivered to time-dated pregnant ewes. INTERVENTIONS:: Fifth generation pulmonary arteries (PA) and lung parenchyma were isolated from control fetal lambs (n = 8) and fetal lambs with PPHN induced by antenatal ductal ligation (n = 9). We studied relaxation responses to various agonists (milrinone, forskolin, prostacyclin, and iloprost, a prostacyclin analog) that increase cAMP in PA after half-maximal constriction with norepinephrine and pretreatment with propranolol +/- indomethacin. Lung protein levels of prostacyclin synthase, IP, AC2, and phosphodiesterase 3A were analyzed by Western blot and cAMP by enzyme-linked immunoassay. MAIN RESULTS:: Milrinone relaxed control and PPHN PA and pretreatment with indomethacin significantly impaired this response. Relaxation to milrinone, prostacyclin, and iloprost were significantly impaired in PA from PPHN lambs. Pretreatment with milrinone markedly enhanced relaxation to prostacyclin and iloprost in PPHN PA, similar to relaxation in control PA. Relaxation to forskolin was similar in control and PPHN PAs indicating normal AC activity. Protein levels of prostacyclin synthase and IP were decreased in PPHN lungs compared with control, but AC2, cAMP, and phosphodiesterase 3A remained unchanged. CONCLUSIONS:: Prostacyclin and iloprost are dilators of PAs from PPHN lambs and their effect is enhanced by milrinone. This combination therapy may be an effective strategy in the management of patients with PPHN.