The peroxidase activity of cytochrome c may play a key role in the release of cytochrome c from the mitochondrial intermembrane space in the intrinsic apoptosis pathway. Induction of the peroxidase activity of cytochrome c is ascribed to partial unfolding and loss of axial co-ordination between the haem Fe and Met80, and is thought to be triggered by interaction of cytochrome c with cardiolipin (diphosphatidylglycerol) in vivo. However, the reaction mechanism for the peroxidase activity of either native or cardiolipin-bound cytochrome c is uncertain. In the present study we analyse the peroxidase activity of human and mouse cytochrome c residue 41 variants and demonstrate that stimulation of peroxidase activity can occur without prior loss of Fe-Met80 co-ordination or partial unfolding. The effects of cardiolipin and mutation of residue 41 are not additive, suggesting that cardiolipin stimulates peroxidase activity by the same mechanism as residue 41 mutation. Consistent with this, mutation of residue 41 did not enhance apoptotic release of cytochrome c from mitochondria. We propose that mutation of residue 41, and interaction with cardiolipin, increase peroxidase activity by altering the 40-57 Ω loop and its hydrogen bond network with the propionate of haem ring A. These changes enhance access of hydrogen peroxide and substrate to the haem.