Inert-gas washout measurements using oxygen, in the lungs of small animals, are complicated by the continuous process of oxygen consumption (Vo2). The multiple-breath nitrogen washout (MBNW) technique uses the alveolar slope to determine measures of ventilation inhomogeneity in the acinar (Sacin) and conducting (Scond) airway regions, as well as overall inhomogeneity, as determined by the lung clearance index (LCI). We hypothesized that measured ventilation inhomogeneity in the mouse lung while it is alive is in fact an artifact due to the high Vo2 in proportion to alveolar gas volume (Va), and not ventilation inhomogeneity per se. In seven male C57BL/6 mice, MBNW was performed alive and postmortem to derive measures with and without the effect of gas exchange, respectively. These results were compared with those obtained from an asymmetric multibranch point mathematical model of the mouse lung. There was no statistical difference in Sacin and LCI between alive and postmortem results (Sacin alive = 0.311 +/- 0.043 ml(-1) and Sacin postmortem = 0.338 +/- 0.032 ml(-1), LCI alive = 7.0 +/- 0.1 and LCI postmortem = 7.0 +/- 0.1). However, there was a significant decrease in Scond from 0.086 +/- 0.005 ml(-1) alive to 0.006 +/- 0.002 ml(-1) postmortem (P <0.01). Model simulations replicated these results. Furthermore, in the model, as Vo2 increased, so did the alveolar slope. These findings suggests that the MBNW measurement of Scond in the mouse lung is confounded by the effect of gas exchange, a result of the high Vo2-to-Va ratio in this small animal, and not due to inhomogeneity within the airways.