Polycarbonate membrane filters were used to fractionate mitochondrial populations depending on their aggregation or association with other subcellular structures. Isolated rat liver mitochondria penetrated through filters which have pore sizes larger than 1 micron. In contrast, mitochondria which were induced to aggregate in vitro by incubation at low pH were retained by the filters and thus could be separated from the single or small aggregates of mitochondria. Use of this membrane filtration method to analyze release of mitochondria from isolated hepatocytes showed that treatment with digitonin at concentrations only sufficient to lyse the plasma membrane did not release mitochondria. Homogenization or sonication following digitonin treatment released 25-50% of the mitochondria, but only a small fraction was intact. A high yield of intact mitochondria was released from digitonin-treated cells by a brief treatment with a low concentration of the proteolytic enzyme nagarse. Thus, this membrane filtration method provides a simple and rapid approach to analyze the extent of mitochondrial aggregation and association with other subcellular structures.