Changes in the spatial distribution of perfusion during acute lung injury and their impact on gas exchange are poorly understood. We tested whether endotoxemia caused topographical differences in perfusion and whether these differences caused meaningful changes in regional ventilation-to-perfusion ratios and gas exchange. Regional ventilation and perfusion were measured in anesthetized, mechanically ventilated pigs in the prone position before and during endotoxemia with the use of aerosolized and intravenous fluorescent microspheres. On average, relative perfusion halved in ventral and cranial lung regions, doubled in caudal lung regions, and increased 1.5-fold in dorsal lung regions during endotoxemia. In contrast, there were no topographical differences in perfusion before endotoxemia and no topographical differences in ventilation at any time point. Consequently, endotoxemia increased regional ventilation-to-perfusion ratios in the caudal-to-cranial and dorsal-to-ventral directions, resulting in end-capillary PO2 values that were significantly lower in dorsal-caudal than ventral-cranial regions. We conclude that there are topographical differences in the pulmonary vascular response to endotoxin that may have important consequences for gas exchange in acute lung injury.