Perfusion of the pulmonary acinus has been shown to be generally homogeneous, but there is a significant component that is heterogeneous. To investigate the contribution of the alveolar septal capillary network to acinar perfusion heterogeneity, the passage of fluorescent dye boluses through the subpleural microcirculation of isolated dog lung lobes was videotaped using fluorescence microscopy. As the videotapes were replayed, dye-dilution curves were recorded from each of the tributary branches of Y-shaped venules that drained single acini. For each Y-shaped venule, the mean appearance time difference between the pair of tributary branches was calculated from the dye curves. When the complex septal capillary networks were derecruited by high positive airway pressure, venular perfusion became proportionally more homogeneous. This result shows that septal capillary resistance and pathlength differences are important contributors to intra-acinar perfusion heterogeneity.