Persistent pulmonary hypertension of the newborn (PPHN) is characterized by endothelial dysfunction and impaired angiogenesis. How rho-kinase activity modulates endothelial function and regulates angiogenesis during normal lung development and in PPHN is unknown. We hypothesized that PPHN increases rho-kinase activity in fetal pulmonary artery endothelial cells (PAECs) and impairs angiogenesis in vitro. Proximal PAECs were harvested from fetal sheep with partial ligation of the ductus arteriosus in utero (PPHN) and age-matched controls. Rho-kinase activity, was measured by rhoA, RhoGTP and P-MYPT-1 protein content. The effects of rho-kinase activity on angiogenesis, eNOS protein expression and NO production were determined in normal and PPHN PAECs. Angiogenesis was assessed by tube formation in vitro with/without Y-27632, (rho-kinase inhibitor), and calpeptin, (rho-kinase activator), in presence/absence of L-NA (NOS inhibitor). RhoA, rho-GTP and P-MYPT-1 protein were increased in PPHN PAECs. Tube formation was reduced by 29% in PPHN PAECs (p<0.001) and increased with Y-27632 treatment in normal and PPHN PAECs with PPHN PAECs achieving similar values to normal PAECs. L-NA inhibited the Y-27632 increase in tube formation in normal but not PPHN PAECs. Calpeptin reduced tube formation in normal and PPHN PAECs. eNOS expression, was reduced by 42% in PPHN PAECs (p<0.01). Y-27632 increased eNOS protein and NO production in normal and PPHN PAECs. Calpeptin decreased eNOS protein only in normal PAECs, but reduced NO production in normal and PPHN PAECs. We conclude that rho-kinase activity is increased in PPHN PAECs, which down-regulates eNOS protein and NO production and impairs angiogenesis in vitro.