The utilization of endogenous stores by rabbit aorta in vitro was measured. In substrate-free medium glycogen disappearance may account for less than 20% of the tissue O2 consumption during incubations of less than 2-3 h. At longer times (or in the presence of glucose) glycogen catabolism is negligible. Calculations from the rate of proteolysis suggest that oxidation of endogenously generated amino acids accounts for less than 7-10% of the oxygen consumption. Furthermore, the presence of amino-oxyacetate, a transaminase inhibitor, did not alter the ATP-ADP ratio. By contrast, measurements of the disappearance of tissue triglyceride indicate that endogenous lipid could meet the fuel requirements of the aorta. Direct measurement of intracellular fatty acid oxidation was obtained by measuring acyl carnitine specific activity and 14CO2 production from [1-14C]palmitate. Fatty acid oxidation could account for at least 90% of the total O2 consumption, and 83% of the fatty acids consumed were derived from endogenous tissue stores. Octanoate was found to inhibit both exogenous and endogenous fatty acid oxidation. These findings may indicate that shorter-chain fatty acids may be preferentially utilized by the aorta.