Recent data show that a strong relation exists in certain cells between mitochondria and caspase activation in apoptosis. We further investigated this relation and tested whether treatment with the permeability transition (PT)-inducing agent atractyloside of Percoll-purified mitochondria released a caspase-processing activity. Following detection of procaspase-11 processing, we further purified this caspase-processing protease and identified it as cathepsin B. The purified cathepsin B, however, was found to be derived from lysosomes which were present as minor contaminants in the mitochondrial preparation. Besides procaspase-11, caspase-1 is also readily processed by cathepsin B. Procaspase-2, -6, -7, -14 are weak substrates and procaspase-3 is a very poor substrate, while procaspase-12 is no substrate at all for cathepsin B. In addition, cathepsin B induces nuclear apoptosis in digitonin-permeabilized cells as well as in isolated nuclei. All newly described activities of cathepsin B, namely processing of caspase zymogens and induction of nuclear apoptosis, are inhibited by the synthetic peptide caspase inhibitors z-VAD.fmk, z-DEVD.fmk and to a lesser extent by Ac-YVAD.cmk.