The atrial Natriuretic Factor (ANF) induces diuresis, natriuresis and vasodilation. ANF relaxation is impaired in adults with primary pulmonary hypertension. The ANF system is also present in newborn piglet pulmonary vessels, but its role in the pathogenesis of pulmonary hypertension (PH) of the newborn in unknown. Therefore, we studied in 1-day-old piglets exposed to hypoxia (FiO2 0.10) for 3 or 14 days to induce PH: (1) ANF release by measuring circulating levels of ANF by radioimmunoassay in pulmonary artery and veins, (2) pulmonary vascular reactivity to ANF using isolated perfused lungs, and (3) binding characteristics by examining the concentration dependence of ANF on membranes from pulmonary arteries (down to 100 μm). ANF circulating levels after 14-day exposure to hypoxia are increased compared to normoxia: 134.9±66.3 vs 59.5±25.7 fmol/ml (n=6; p<0.001). The magnitude of ANF dilator response is greatly diminished after 3-day hypoxia (n=3; p<0.05) and further abolished after 14-day (n=3; p<0.05). Saturation studies (n=2-3) reveal that the population of ANF receptors is significantly diminished in hypoxia after 3-day (1508±198 vs 2950±248 fmol/mg; p<0.01), but does not reach significance after 14-day (993±333 vs 1511±282 fmol/mg) compared to their respective normoxic control. These results suggest that hypoxia increases ANF synthesis or reduces clearance followed by a downregulation of ANF receptors leading to a diminished vasodilator response of the pulmonary vessels. Throughout these effects, the ANF system could play a major role in the pathogenesis of PPHN.