Acetoacetate provision to Ca(2+)-loaded liver mitochondria (less than 40 micrograms-ion Ca2+ x g protein-1), supplied with 2 mM Pi and 2-oxoglutarate as substrate, was found to prevent the mitochondrial deenergization and Ca2+ release induced by either rotenone during aerobic incubations or by O2 deprivation. Under the latter condition, the acetoacetate-promoted Ca2+ retention was entirely supported by ATP produced anaerobically at the succinylthiokinase step of the tricarboxylic acid cycle and was therefore abolished by addition of oligomycin. Surprisingly, oligomycin was also found to trigger Ca2+ release in rotenone-inhibited mitochondria in the presence of acetoacetate under aerobic conditions, unless a Pi acceptor was supplied. ADP deprivation at the succinylthiokinase step is likely to be involved. As estimated from rates of succinate production in O2-deprived mitochondria or from respiration rates in rotenone-inhibited mitochondria at supramaximal acetoacetate concentrations (above 1.2 mM) in the presence of a Pi acceptor, ATP production by substrate-level phosphorylation was close to 10 mumol.g protein-1.min-1 and appeared to be limited by rates of ketone body transport across the inner membrane. The rates of anaerobic energy production obtained by coupling 2-oxoglutarate oxidation to acetoacetate reduction were markedly higher than those obtained by reactions involved in the anaerobic metabolism of amino acids, simulated by providing 2-oxoglutarate and malate to mitochondria. Energy production was limited by rates of oxidant equivalent generation under the latter condition. Our data suggest that acetoacetate could effectively contribute to sustaining anaerobic energy production from endogenous substrates in liver tissue.