Abstract (1) The mitoplasts were prepared from bullfrog ( Rana catesbeiana) liver mitochondria by treatment with digitonin and were then separated into the matrix and inner membrane fractions. The matrix fraction thus obtained was free of lysosomal contaminations and exhibited a distinct proteinase activity. (2) pH dependency of the matrix proteinase activity measured in the presence and absence of iodoacetamide revealed that the matrix contained at least two kinds of proteinase, a major alkaline thiol proteinase having an optimal pH at 8.5 and a minor neutral proteinase having an optimal pH at 7.5. (3) The major matrix proteinase activity was strongly inhibited by leupeptin, chymostatin, antipain and E64-C, an inhibitor of Ca 2+-dependent thiol proteinase, while it was scarcely affected by diethylpyrocarbonate. The activity was also inhibited by DTNB and p-chloromercuribenzoate. (4) Addition of hydrocarbon compounds such as ethylene glycol, glycerol, Triton X-100 and poly(ethylene glycol) to the reaction mixture was found to decrease the matrix proteinase activity. (5) Neither cytochrome c nor glutamate dehydrogenase was hydrolyzed when subjected to the matrix proteinase activity in vitro. On the other hand, cytochrome c oxidase was effectively hydrolyzed, and the enzyme associated with the mitochondrial innermembrane fragments was partially hydrolyzed by the major matrix proteinase activity.