Several studies have proposed that the antibiotic minocycline (MC) has cytoprotective activities. Nevertheless, when cells have been exposed to MC at micromolar concentrations, detrimental effects have been also observed. Despite the known inhibitory activity of MC on ATP synthesis and the Ca2+ retention capacity of isolated rat liver and brain mitochondria, the underlying mechanism is still debated. Here, we present further arguments supporting our concept that MC acting on rat liver mitochondria suspended in KCl medium permeabilizes the inner membrane. Supplementation of the medium with cytochrome c and NAD+ strongly enhanced the respiration of MC-treated mitochondria, thus partly preventing or reversing the inhibitory effect of MC on state 3 or uncoupled respiration. These results indicate that MC produced depletion of mitochondrial cytochrome c and NAD+, thus impairing mitochondrial respiration. In addition, NADH oxidation by alamethicin-permeabilized mitochondria supplemented with cytochrome c was insensitive to 200 μm MC, arguing against direct impairment of respiratory chain complexes by MC. Finally, a surprising feature of MC was its accumulation or binding by intact rat liver mitochondria, but not by mitochondria permeabilized with alamethicin or disrupted by freezing and thawing.