We previously reported the construction of an overexpression system of Candida tropicalis peroxisomal catalase (CTC) gene in the Saccharomyces cerevisiae cytoplasm using the GAL7 promoter (Kinoshita et al., Appl. Microbiol. Biotechnol., 40 : 682-686, 1994). To study the mechanism of the subcellular localization of peroxisomal catalase which is composed of four identical subunits and four heme molecules, the system was improved by changing the promoter used in order to express the catalase subunit accompanying the proliferation of peroxisomes in S. cerevisiae cells (S. cerevisiae Delta A Delta T strain), in which catalase A and catalase T genes were disrupted. On native-PAGE, besides the functional tetrameric form (220 kDa), other larger multimeric forms of recombinant CTC were observed in the cell-free extract. It was clearly demonstrated that only the tetrameric form, among the various forms, contained heme and exhibited catalase activity. Subcellular fractionation and protease protection experiments indicated that only the functionally active tetrameric form was present inside the peroxisomes even under conditions of overexpression of the catalase subunit, in which different multimeric forms including the active tetrameric form were produced outside the peroxisomes. The results indicated that C. tropicalis catalase subunit and heme stoichiometrically localize even in S. cerevisiae peroxisomes, suggesting a common regulatory role of peroxisomes themselves for the transport of the oligomeric form of catalase beyond yeast species.