The spatial distribution of pulmonary blood flow is increasingly heterogeneous as progressively smaller lung regions are examined. To determine the extent of perfusion heterogeneity at the level of gas exchange, we studied blood dow distributions in rat lungs by using an imaging cryomicrotome. Approximately 150,000 fluorescent 15-mu m-diameter microspheres were injected into tail veins of five awake rats. The rats were heavily anesthetized; the lungs were removed, filled with an optimal cutting tissue compound, and frozen; and the spatial location of every microsphere was determined. The data were mathematically dissected with the use of an unbiased random sampling method. The coefficients of variation of microsphere distributions were determined at varying sampling volumes. Perfusion heterogeneity increased linearly on a log-log plot of coefficient of variation vs. volume, down to the smallest sampling size of 0.53 mm(3). The average fractal dimension, a scale-independent measure of perfusion distribution, was 1.2. This value is similar to that of other larger species such as dogs, pigs, and horses; Pulmonary perfusion heterogeneity increases continuously and remains fractal down to the acinar level. Despite the large degree of perfusion heterogeneity at the acinar level, gases are efficiently exchanged.