A biochemical and morphological study of an electron-transport system associated with the outer membrane of liver mitochondria is presented. Rat-liver mitochondria catalyze the oxidation of exogenous NADH by cytochrome c or ferricyanide, a reaction insensitive to respiratory chain inhibitors like antimycin A, amytal, and rotenone, and not coupled to phosphorylation. This "external" NADH-cytochrome c reductase activity resembles the microsomal NADH-cytochrome c reductase system stereochemically but differs from the respiratory chain-linked NADH dehydrogenase. Enzyme distribution data show that microsomal contamination cannot account for this activity. A procedure involving swelling and shrinking of mitochondria, followed by sonication and density gradient centrifugation, separates two subfractions: one containing the respiratory chain components and the other containing the rotenone-insensitive NADH-cytochrome c reductase system. Morphological evidence supports the conclusion that the former subfraction consists of mitochondria devoid of outer membrane, while the latter represents derivatives of the outer membrane. The data indicate that the electron-transport system associated with the mitochondrial outer membrane involves catalytic components similar to or identical with the microsomal NADH-cytochrome b5 reductase and cytochrome b5. Electron microscopy revealed distinct differences between the heavy and light subfractions, with the heavy subfraction containing large vesicles bounded by a single membrane and the light subfraction containing smaller, flattened vesicles. These findings suggest that the rotenone-insensitive NADH-cytochrome c reductase system is associated with the outer mitochondrial membrane.A biochemical and morphological study of an electron-transport system associated with the outer membrane of liver mitochondria is presented. Rat-liver mitochondria catalyze the oxidation of exogenous NADH by cytochrome c or ferricyanide, a reaction insensitive to respiratory chain inhibitors like antimycin A, amytal, and rotenone, and not coupled to phosphorylation. This "external" NADH-cytochrome c reductase activity resembles the microsomal NADH-cytochrome c reductase system stereochemically but differs from the respiratory chain-linked NADH dehydrogenase. Enzyme distribution data show that microsomal contamination cannot account for this activity. A procedure involving swelling and shrinking of mitochondria, followed by sonication and density gradient centrifugation, separates two subfractions: one containing the respiratory chain components and the other containing the rotenone-insensitive NADH-cytochrome c reductase system. Morphological evidence supports the conclusion that the former subfraction consists of mitochondria devoid of outer membrane, while the latter represents derivatives of the outer membrane. The data indicate that the electron-transport system associated with the mitochondrial outer membrane involves catalytic components similar to or identical with the microsomal NADH-cytochrome b5 reductase and cytochrome b5. Electron microscopy revealed distinct differences between the heavy and light subfractions, with the heavy subfraction containing large vesicles bounded by a single membrane and the light subfraction containing smaller, flattened vesicles. These findings suggest that the rotenone-insensitive NADH-cytochrome c reductase system is associated with the outer mitochondrial membrane.