Acetoacetyl-CoA thiolase (Thiolase I) and 3-ketoacyl-CoA thiolase (Thiolase III) found in peroxisomes of an n-alkane-utilizing yeast, Candida tropicalis pK 233, were each purified to homogeneity by successive column chromatographies. Thiolase I was composed of six identical subunits whose molecular masses were 41, 000 Da, and Thiolase III was a homodimer composed of 43, 000 Da subunits. The results of limited proteolysis of the respective thiolases indicated that they were quite different in peptide components. Furthermore, these enzymes were immunochemically distinguishable. The kinetic studies showed that the substrates with long chains were degraded exclusively by Thiolase III, while acetoacetyl-CoA was degraded preferentially by Thiolase I. Thus, in the yeast, the complete degradation of fatty acids is suggested to be carried out efficiently in peroxisomes.