A study is presented on the interaction of ceramide with the respiratory chain of rat heart mitochondria, and a comparison is made between the effects elicited by short- and long-chain ceramides. N-Acetylsphingosine (C₂-ceramide) and N-palmitoylsphingosine (C₁₆-ceramide) inhibited to the same extent the pyruvate+malate-dependent oxygen consumption. Succinate-supported respiration was also inhibited by ceramides, but this activity was substantially restored upon the addition of cytochrome c, which, on the contrary, was ineffective toward the ceramide-inhibited NADH-linked substrate oxidation. Direct measurements showed that short- and long-chain ceramides caused a large release of cytochrome c from mitochondria. The ceramide-dependent inhibition of pyruvate+malate and succinate oxidation caused reactive oxygen species to be produced at the level of either complex I or complex III. The activity of the cytochrome c oxidase, measured as ascorbate/TMPD oxidase activity, was significantly stimulated and inhibited by C₂- and C₁₆-ceramide, respectively. Similar effects were observed on the activity of the individual respiratory complexes isolated from bovine heart. Short- and long-chain ceramides had definitely different effects on the mitochondrial membrane potential. C₂-ceramide caused an almost complete collapse of the respiration-dependent membrane potential, whereas C₁₆-ceramide had a negligible effect. Similar results were obtained when the potential was generated in liposome-reconstituted complex III respiring at the steady-state. Furthermore, C₂-ceramide caused a drop of the membrane potential generated by ATP hydrolysis instead of respiration, whereas C₁₆-ceramide did not. Finally, only short-chain ceramides inhibited markedly the reactive oxygen species generation associated with membrane potential-dependent reverse electron flow from succinate to complex I. The emerging indication is that the short-chain ceramide-dependent collapse of membrane potential is a consequence of their ability to perturb the membrane structure, leading to an unspecific increase of its permeability.