Morphological changes are observed in mitochondria early in the course of acetaminophen (APAP) hepatotoxicity. In order to determine if functional deficits also occur, this study examined the effect of APAP, in vivo and in vitro, on mitochondrial respiration in fasted, male CD-1 mice (3–4 months old). After a hepatotoxic dose of APAP (600 mg/kg, po), when glutamate was used as the respiratory substrate, state 3 respiration (ADP-stimulated) was inhibited and this was reflected in a decreased respiratory control ratio (RCR). In contrast, when succinate was the respiratory substrate, the decreased RCR was reflective of an increase in state 4 (resting) respiration. There was no detectable effect after a nonhepatotoxic dose of APAP (300 mg/kg, po). These APAP-induced respiratory effects and hepatotoxicity were prevented by piperonyl butoxide pretreatment, and were absent in 1- and 2-month-old mice, which are resistant to APAP-induced damage. Since the APAP-induced inhibition of mitochondrial respiration, in vivo, correlated with age-related and piperonyl butoxide-dependent differences in toxicity, the data suggest that the in vivo effects result, at least in part, from a mixed-function oxidase generated metabolite. In vitro, both state 3 and state 4 respiration, as well as the RCR, were inhibited by APAP in a concentration-dependent manner with glutamate as substrate. However, no effects were observed with succinate as substrate, thereby contrasting with results obtained following in vivo exposure. Therefore the in vitro effects of APAP are different from those observed in vivo and may result from a direct insult of the parent compound. These studies suggest that early alterations in mitochondrial function may be mechanistically important in APAP hepatotoxicity.