In the present report we describe a method for the complete purification of native sterol carrier protein 2/3-oxoacyl-CoA thiolase (SCP-2/thiolase) from normal rat liver peroxisomes. The isolation procedure is based on the alteration in chromatographic properties of the enzyme in the presence of low concentrations of CoA. The purified preparation of SCP-2/thiolase consisted of 58- and 46-kDa polypeptides. Peroxisomes prepared freshly from normal rat liver contained three SCP-2/thiolase isoforms, separable by conventional chromatography. Immunochemical, molecular sieving, and chemical cross-linking experiments indicated that these isoforms represent thiolytically active homo- and heterodimeric combinations of the 46- and 58-kDa subunits (2 x 58, 58-46, and 2 x 46-kDa proteins).