Rationale: Sepsis significantly alters skeletal muscle mitochondrial function, but the mechanisms responsible for this abnormality are unknown. Objectives: We postulated that endotoxin elicits specific changes in electron transport chain proteins that produce derangements in mitochondrial function. To examine this issue, we compared the effects of endotoxin-induced sepsis on mitochondrial ATP (adenosine triphosphate) formation and electron transport chain protein composition. Methods and Measurements: Diaphragm mitochondrial oxygen consumption and mitochondrial NADH (nicotinamide adenine dinucleotide, reduced form) oxidase assays were measured in control rats (n=13) and rats given endotoxin (8 mg/kg/d) for 12 (n=14), 24 (n=14), 36 (n=14), and 48 hours (n=13). Electron transport chain subunits from Complexes I, III, IV and V were isolated using Blue Native polyacrylamide gel electrophoresis techniques (BN-PAGE). Main Results: Endotoxin administration: (a) elicited large reductions in mitochondrial oxygen consumption (e.g. 201 ± 3.9 SE natoms O/min/mg for controls and 101 ± 4.5 SE natoms O/min/mg after 48 hours endotoxin, p<0.001), in NADH oxidase activity (p<0.002), and in uncoupled respiration (p<0.001); and, (b) induced selective reductions in 2 subunits of Complex I, 3 subunits of Complex III, 1 subunit of Complex IV, and 1 subunit of Complex V. The time course of depletion of protein subunits mirrored alterations in oxygen consumption. Conclusions: Our data indicate that endotoxin selectively depletes critical components of the electron transport chain that diminishes electron flow, reduces proton pumping and decreases ATP formation.