The effects of diltiazem on fatty acid metabolism were measured in the isolated perfused rat liver and in isolated mitochondria. In the perfused rat liver diltiazem inhibited oxygen uptake and ketogenesis from endogenous substrates. Ketogenesis from exogenously supplied palmitate was also inhibited. The beta-hydroxybutyrate/acetoacetate ratio in the presence of palmitate alone was equal to 3.2. When the fatty acid and diltiazem were present simultaneously this ratio was decreased to 0.93, suggesting that, in spite of the inhibition of oxygen uptake, the respiratory chain was not rate limiting for the oxidation of the reducing equivalents coming from beta-oxidation. In experiments with isolated mitochondria, incubated in the presence of all intermediates of the Krebs cycle, pyruvate or glutamate, no significant inhibition of oxygen uptake by diltiazem was detected. Inhibition of oxygen uptake in isolated mitochondria was found only when palmitoyl CoA was the source of the reducing equivalents. It was concluded that a direct effect on beta-oxidation may be a major cause for the inhibition of oxygen uptake caused by diltiazem in the perfused liver.