We studied the in vitro effect of E. coli endotoxin on the oxidation of palmitate, palmitoyl CoA, and lactate by canine heart homogenate. Heart homogenates were incubated in calcium-free Krebs-Ringer-phosphate buffer in the presence of a 14C-labeled substrate. Oxidation of the individual substrate was calculated from the rate of 14CO2 production. The rate of oxidation of palmitate, palmitoyl CoA, and lactate was proportionally inhibited by increasing amounts (80-800 microgram) of endotoxin. The decrease in substrate oxidation could be mimicked by adding calcium chloride to the tissue preparation, and could be effectively prevented by the chelating agent, EDTA. Ionic calcium was released from tissue stores during incubation of the tissue preparation with endotoxin. These findings demonstrate that E. coli endotoxin inhibits substrate oxidation by heart homogenates when incubated under in vitro conditions. The data also suggest that the inhibition may be mediated by ionic calcium released from the tissue in response to the action of endotoxin.