Detailed respiration studies on isolated liver mitochondria from streptozotocin-induced diabetic Sprague-Dawley rats revealed a disease-associated decrease in the ADP/O ratio, a marker for mitochondrial ability to couple the consumption of oxygen to the phosphorylation of ADP. This decrease was observed following induction of respiration with glutamate/malate, succinate, or duroquinol, which enter the electron transport chain selectively at complexes I (NADH dehydrogenase), II (succinate dehydrogenase), or III (cytochrome bc1 complex), respectively. These data, coupled with studies using respiratory inhibitors (most importantly antimycin A and myxothiazol), localize at least a portion of this defect to a single site within the electron transport chain (center P in the Q-cycle portion of complex III). These results suggest that liver mitochondria from diabetic animals may generate increased levels of reactive oxygen species at the portion of the electron transport chain already established as the major site of mitochondrial free radical generation. The reduction in the ADP/O ratio occurred in mitochondria that do not have overt defects in the respiratory control ratio or in State 3 and State 4 respiration. The data in this paper suggest that defects in center P of the electron transport chain likely increase mitochondrial exposure to oxidants in the diabetic. This data may partially explain the evidence of altered exposure and/or response to reactive species in mitochondria from diabetics. This work thus provides further clues to the interaction between oxidative stress and diabetes-associated mitochondrial dysfunction.