Cardiolipin plays an important role in mitochondrial membrane structure and function. We have recently reported a decrease in the cytochrome c oxidase activity in heart mitochondria from hypothyroid rats (G. Paradies et al. (1993) Arch. Biochem Biophys. 307, 91-95). A possible involvement of cardiolipin in such a decrease has been proposed. The aim of this work was to test our earlier proposal. We have investigated whether addition of exogenous cardiolipin to hypothyroid mitochondria is able to reverse, in situ, their decreased cytochrome oxidase activity. The method of fusion of liposomes with mitochondria developed by Hackenbrock (Hackenbrock and Chazotte (1986) Methods Enzymol. 125, 35-45) was employed in order to enrich the mitochondrial cardiolipin content. We demonstrate that the decreased activity of this enzyme complex in heart mitochondria from hypothyroid rats can be completely restored to the level of control rats by exogenously added cardiolipin but not by other phospholipids. These data provide strong evidence for the involvement of cardiolipin in the thyroid hormone induced changes of mitochondrial cytochrome oxidase activity.