We previously reported differential regulation of hypoxia-inducible factors (HIF-1, -2 and -3) mRNA in dog lungs during normal maturation and post-pneumonectomy (PNX) compensatory growth in the overt absence of hypoxia (Zhang et al., Am J Physiol Lung Cell Mol Physiol, 290:L880-889, 2006). To test the hypothesis that lung expansion activates HIF signaling, we replaced the right lung of 6 adult foxhounds with inflated custom-shaped silicone prosthesis to keep the mediastinum in the midline and minimize lateral expansion of the remaining lung. After 3 wk of recovery and stabilization of perfusion, the prosthesis was acutely deflated in 3 animals, causing the remaining lung to expand by 114%. In 3 other animals the prosthesis remained inflated. Three days following deflation, we observed significant elevation in the mRNA and nuclear protein levels of HIF-1 (~60%) as well as activation of its transcriptional regulator the serine/threonine protein kinase B (phospho-Akt/total Akt ratio, 124%) and the mRNA and protein levels of its downstream targets, erythropoietin receptor (EPO-R, 71-183%) and vascular endothelial growth factor (VEGF, 33-58%), compared to the pre-PNX control lung from the same animal. The mRNA of HIF-2, HIF-3 and VEGF receptors did not change with acute deflation. We conclude that in vivo lung expansion by post-PNX deflation of space-occupying prosthesis elicits coordinated activation of HIF-1 signaling in adult lungs. This pathway could play an important role in mediating lung growth and remodeling during maturation and post-PNX compensation.