Previous studies indicate that ATP formation by the electron transport chain is impaired in sepsis. However, it is not known if sepsis affects the mitochondrial ATP transport system. We hypothesized that sepsis inactivates the mitochondrial creatine kinase-high energy phosphate transport system. To examine this issue, we assessed the effects of endotoxin administration on mitochondrial membrane-bound creatine kinase, an important trans-mitochondrial ATP transport system. Diaphragms and hearts were isolated from control (n=12) and endotoxin-treated (8 mg/kg/d, n=13) rats after pentobarbital anesthesia. Mitochondria were isolated using techniques which allow evaluation of the functional coupling of mitochondrial creatine kinase (MtCK) activity to oxidative phosphorylation. MtCK functional activity was established by: (1) determining ATP/creatine-stimulated oxygen consumption and (2) assessing total creatine kinase activity in mitochondria using an enzyme-linked assay. MtCK protein content was examined using Western blots. Endotoxin markedly reduced diaphragm and cardiac MtCK activity, as determined both by ATP/creatine-stimulated oxygen consumption and by the enzyme-linked assay (e.g. ATP/creatine-stimulated mitochondrial respiration was 173.8±7.3, 60.5±9.3, 210.7±18.9, 67.9±7.3 natoms O/min/mg in diaphragm control, diaphragm septic, cardiac control, and cardiac septic samples, respectively, p<0.001 for each tissue comparison). Endotoxin also reduced diaphragm and cardiac MtCK protein levels (e.g. protein levels declined by 39.5% in diaphragm mitochondria and by 44.2% in cardiac mitochondria, p<0.001 and p=0.009, respectively, comparing sepsis to control conditions). Our data indicate that endotoxin markedly impairs the MtCK-ATP transporter system; this phenomenon may have significant effects on diaphragm and cardiac function.