Mitochondrial processing peptidase (MPP), consisting of alpha and beta subunits, recognizes a large variety of N-terminal extension peptides of mitochondrial precursor proteins, and generally cleaves a single site of the peptide including arginine at the -2 position (P-2). We obtained evidence that Glu(191) and Asp(195) of rat beta subunit interact with P-2 arginine of precursor protein through ionic and hydrogen bonds, respectively, using recombinant MPP. Mutation to alanines at Glu(191) and Asp(195) reduced processing activity toward precursors with P-2 arginine, but resulted in no loss of activity toward P-2 alanine precursors. Charge-complementary mutation demonstrated that MPP variants with beta Arg(191) exhibited compensatory processing activity for the precursor with acidic residue at the P-2 position. Thus, Glu(191) and Asp(195) are substrate-binding sites required for cleavage of extension peptides through interaction with P-2 arginine.