Previously, we reported that the major site of pulmonary vascular resistance in fetal lambs occurred in the middle region defined by vascular occlusion, and that this region exhibited the greatest decrease upon ventilation with O2. To assess the relative individual contributions of ventilation and oxygenation to this decrease, we determined the distribution of pressures across the pulmonary circulation in isolated perfused lungs from 20 fetal lambs (131–137 d gestation) by inflow and outflow vascular occlusions. A membrane oxygenator was included in the extracorporeal circuit to control the Po1at 4 kPa (30 torr) in the unventilated fetal lungs. Half of the fetal lungs were ventilated first without changing the initial gas tensions, and the others were oxygenated first by changing the initial gas tensions to a hyperoxic mixture [Po2= 26.6 kPa (200 torr)] without ventilation. Finally, both groups of lungs were ventilated and oxygenated. In addition, indomethacin was added to the perfusate (0.112 mM, or 40 μg/mL) in half of the preparations in each group to determine the effect of prostaglandins on the distribution of pressures during these conditions. The decrease in the total pulmonary vascular resistance with ventilation and/or oxygenation was primarily due to changes in the middle pressure gradient (ΔPm). In fetal lungs without indomethacin, ventilation without oxygenation reduced ΔPm from 6.1 ± 0.8 to 2.5 ±1.0 kPa, or 74% of the total ventilation- and oxygenation-induced decrease in ΔPm (final value = 1.2 ± 0.6 kPa). In contrast, oxygenation without ventilation produced a decrease in ΔPm from 5.5 ± 0.7 to 3.8 ± 0.5 kPa, only 40% of the total decrease in ΔPm (1.2 ± 0.4 kPa). Furthermore, in fetal lungs with indomethacin, only oxygenation first caused a reduction in the resistance of the middle region, suggesting that dilator prostaglandins are not involved in the response to increased oxygen. We conclude that recruitment and/or distension of the small pulmonary vessels functionally located in the middle region by the mechanical effect of ventilation is dependent on dilator cyclooxygenase products, and that this mechanical effect is a major factor involved in the decrease in pulmonary vascular resistance occurring at birth.