We studied the physiological role of flow through pulmonary arterioles in CO2 gas exchange. We established human pulmonary arteriolar endothelial cells (HPAoEC). The cells demonstrated marked immunocytochemical staining of PECAM-1, VEGF R2, ACE-1, and CA type IV on their cell surface. Ten seconds shear stress stimulation caused the co-release of H+ and ATP via the activation of F1/FO ATP synthase on the HPAoEC. F1/FO ATP synthase was immunocytochemically observed on the cell surface of non-permeabilized HPAoEC. In the shear stress-loaded HPAoEC culture media supernatant, ATPase activity increased in a time-dependent manner. The HPAoEC were strongly stained for NTPDase 1, which partially co-localized with purinergic P2Y1. The purinergic P2Y1 receptor agonist UTP (10−6 M) significantly potentiated the shear stress-induced increase in ATPase activity in the culture medium supernatant. Ten seconds shear stress stimulation also produced stress strength-dependent CO2 gas excretion from the HPAoEC, which was significantly reduced by the inhibition of F1/FO ATP synthase or CA IV on the endothelial cell (EC) surface. In conclusion, we have proposed a new concept of CO2 exchange in the human lung, flow-mediated F1/FO ATP synthase-dependent H+ secretion, resulting in the facilitation of a dehydration reaction involving in plasma and the excretion of CO2 gas from arteriolar ECs. J. Cell. Physiol. 227: 2059–2068, 2012. © 2011 Wiley Periodicals, Inc.