This study investigates the localization of soluble and particulate enzymes in rat liver mitochondria using digitonin fractionation. The mitochondria are separated into three fractions: inner membrane plus matrix, outer membrane, and a soluble fraction containing enzymes between the membranes and some outer membrane. Monoamine oxidase, kynurenine hydroxylase, and rotenone-insensitive NADH-cytochrome c reductase are primarily found in the outer membrane fraction. Succinate-cytochrome c reductase, succinate dehydrogenase, cytochrome oxidase, β-hydroxybutyrate dehydrogenase, α-ketoglutarate dehydrogenase, lipoamide dehydrogenase, NAD- and NADH-isocitrate dehydrogenase, glutamate dehydrogenase, aspartate aminotransferase, and ornithine transcarbamoylase are found in the inner membrane-matrix fraction. Nucleoside diphosphokinase is found in both the outer membrane and soluble fractions, suggesting dual localization. Adenylate kinase is found entirely in the soluble fraction and is released at a lower digitonin concentration than the outer membrane, indicating it is localized between the two membranes. The inner membrane-matrix fraction is further separated into inner membrane and matrix using the nonionic detergent Lubrol, and this separation is used to calculate the relative protein content of mitochondrial components. The inner membrane-matrix fraction retains morphological and biochemical integrity and exhibits respiratory control when assayed in a sucrose-mannitol medium containing EDTA. The study also discusses the use of Lubrol for activating mitochondrial enzymes and the distribution of glucose-6-phosphatase, which is found primarily in microsomal membranes rather than the outer mitochondrial membrane.This study investigates the localization of soluble and particulate enzymes in rat liver mitochondria using digitonin fractionation. The mitochondria are separated into three fractions: inner membrane plus matrix, outer membrane, and a soluble fraction containing enzymes between the membranes and some outer membrane. Monoamine oxidase, kynurenine hydroxylase, and rotenone-insensitive NADH-cytochrome c reductase are primarily found in the outer membrane fraction. Succinate-cytochrome c reductase, succinate dehydrogenase, cytochrome oxidase, β-hydroxybutyrate dehydrogenase, α-ketoglutarate dehydrogenase, lipoamide dehydrogenase, NAD- and NADH-isocitrate dehydrogenase, glutamate dehydrogenase, aspartate aminotransferase, and ornithine transcarbamoylase are found in the inner membrane-matrix fraction. Nucleoside diphosphokinase is found in both the outer membrane and soluble fractions, suggesting dual localization. Adenylate kinase is found entirely in the soluble fraction and is released at a lower digitonin concentration than the outer membrane, indicating it is localized between the two membranes. The inner membrane-matrix fraction is further separated into inner membrane and matrix using the nonionic detergent Lubrol, and this separation is used to calculate the relative protein content of mitochondrial components. The inner membrane-matrix fraction retains morphological and biochemical integrity and exhibits respiratory control when assayed in a sucrose-mannitol medium containing EDTA. The study also discusses the use of Lubrol for activating mitochondrial enzymes and the distribution of glucose-6-phosphatase, which is found primarily in microsomal membranes rather than the outer mitochondrial membrane.