Lung aeration stimulates the increase in pulmonary blood flow (PBF) at birth, but spatial relationships between PBF and lung aeration and the role of increased oxygenation remains unclear. Using simultaneous phase-contrast X-ray imaging and angiography, we have investigated the separate roles of lung aeration and increased oxygenation in PBF changes at birth using near-term (30d gestation) rabbit kittens (n = 18). Rabbits were imaged pre-ventilation, then the right lung was ventilated with 100 nitrogen (N2 ), air or 100 O2 (oxygen), before all kittens were switched to ventilation in air, followed by ventilation of both lungs using air. Unilateral ventilation of the right lung with 100 N2 significantly increased heart rate (from 69.4 +/- 4.9 to 93.0 +/- 15.0 bpm), the diameters of both left and right pulmonary axial arteries, number of visible vessels in both left and right lungs, relative PBF index in both PAs and reduced bolus transit time for both left and right axial arteries (from 1.34 +/- 0.39 s and 1.81 +/- 0.43 s to 0.52 +/- 0.17 s and 0.89 +/- 0.21 s in the left and right axial arteries, respectively). Similar changes were observed with 100 oxygen, but increases in visible vessel number and vessel diameter of the axial arteries were greater in the ventilated right lung during unilateral ventilation. These findings confirm that PBF increase at birth is not spatially related to lung aeration and that the increase in PBF to unventilated regions is unrelated to oxygenation, although oxygen can potentiate this increase. This article is protected by copyright. All rights reserved.