In previous studies using isolated perfused rabbit lungs, an O2 deficit measured by an alveolar gas-to-end capillary blood P(O2) difference (A-aD(O2)) was absent at blood flows (Q) consistent with severe exercise. Thus factors such as VA/Q heterogeneity, shunt and diffusion limitation that contribute to an O2 deficit in vivo were absent. Here we attempted to increase diffusion limitation to O2 transport by reducing the equilibration coefficient D/(betaQ), the ratio of the diffusing capacity (D) to the product of Q and the capacitance coefficient (beta, the slope of the blood O2 content-P(O2) curve). First, we used hypoxic (10% O2) ventilation in conjunction with a low PV(O2) (approximately 25 mmHg) because beta is largest in this region of the O2 dissociation curve. Second, we increased beta by decreasing blood P(CO2) which shifts the O2 dissociation curve to the left (Bohr effect). Third, we increased Q to three times control to reduce D/Q. CO diffusing capacity was measured as a function of blood flow and blood P(O2). A deficit in O2 transport as measured by a significant A-aD(O2) was measured only under conditions of hypoxia and high blood flow. The measured O2 deficit matched the predictions from the equilibration coefficients D/(betaQ) based on measurements of beta, D and Q.