Alterations in liver mitochondria as consequence of rat poisoning with carbon tetrachloride (CCl4) have been reported over many years, but the mechanisms responsible for causing such damage are still largely unknown. Isolated rat liver mitochondria incubated under hypoxic conditions with succinate and ADP were found able to activate CCl4 to a free-radical species identified as trichloromethyl free radical (CCl3) by e.s.r. spectroscopy coupled with the spin-trapping technique. The incubation of mitochondria in air decreased free-radical production, indicating that a reductive reaction was involved in the activation of CCl4. However, in contrast with liver microsomes (microsomal fractions), mitochondria did not require the presence of NADPH, and the process was not significantly influenced by inhibitors of cytochrome P-450. The addition of inhibitors of the respiratory chain such as antimycin A and KCN decreased free-radical formation by only 30%, whereas rotenone displayed a greater effect (approx. 84% inhibition), but only when preincubated for 15 min with mitochondria not supplemented with succinate. These findings suggest that the mitochondrial electron-transport chain is responsible for the activation of CCl4. A conjugated-diene band was observed in the lipids extracted from mitochondria incubated with CCl4 under anaerobic conditions, indicating that stimulation of lipid peroxidation was occurring as a result of the formation of free-radical species.