No mitochondrial processing peptidase (MPP) activity is detected in crystalline bovine heart mitochondrial cytochrome bc1 complex, which possesses full electron transfer activity. However, when the complex is treated with increasing concentrations of Triton X-100 at 37 degreesC, the electron transfer activity decreases, whereas peptidase activity increases. Maximum MPP activity is obtained when the electron transfer activity in the complex is completely inactivated with 1.5 mM of Triton X-100. This result supports our suggestion that the lack of MPP activity in the mammalian cytochrome bc1 complex is because of binding of an inhibitor polypeptide to the active site of MPP located at the interface of core subunits I and II. This suggestion is based on the three-dimensional structural information for the bc1 complex and the sequence homology between subunits of MPP and the core subunits of the beef complex. Triton X-100, at concentrations that disrupt the structural integrity of the bc1 complex as indicated by the loss of its electron transfer activity, weakens the binding of inhibitor polypeptide to the active site of MPP in core subunits, thus activating MPP. The Triton X-100-activated MPP is pH-, buffer system-, ionic strength-, and temperature-dependent. Maximum activity is observed with an assay mixture containing 15 mM Tris-HCl buffer at neutral pH (6.5-8.5) and at 37 degreesC. Activated MPP is completely inhibited by metal ion chelators such as EDTA and o-phenanthroline and partially inhibited by myxothiazol (58%), ferricyanide (28%), and dithiothreitol (81%). The metal ion chelator-inhibited activity can be partially restored by the addition of divalent cations such as Zn2+ (68%), Mg2+ (44%), Mn2+ (54%), Co2+ (62%), and Fe2+ (92%), indicating that metal ion is required for MPP activity. The cleavage site specificity of activated MPP depends more on the length of amino acid sequence from the mature protein portion and less on the presequence portion, when a synthetic peptide composed of NH2-terminal residues of a mature protein and the COOH-terminal residues of its presequence is used as a substrate.