Abstract: In the present work, the recorded kinetics of chemiluminescence enhanced by coumarin 334 indicated that the complexes of cytochrome c (Cyt) with anionic lipids (Cyt-AL), such as cardiolipin (CL) and phosphatidic acid (PA), when added to mitochondrial and cytoplasmic membranes, are capable of catalysing reactions of lipid free radical formation. The effect of Cyt-CL complex was studied on mitochondria isolated from the liver of male Wistar rats and male outbred mice, as well as on erythrocytes isolated from the blood of male Wistar rats. It was shown that complex Cyt-PA exerts a peroxidase effect similar to the effect produced by Cyt-CL. Upon exposure to Cyt-CL, the formation of free radicals was more prominent in mitochondrial ghosts (mitochondria subjected to freeze-thawing and washed by several precipitation–resuspension cycles) than in intact mitochondria. This suggests that Cyt-CL does not pass through biological membranes: even if it penetrates into them, it probably gets stuck in the phospholipid bilayer. Thus, the previously discovered cytotoxic effect of Cyt-CL on cancer cells is most likely due to peroxidation of the cytoplasmic membrane, but not of the inner membrane of mitochondria.