The cytosolic and mitochondrial forms of serine hydroxymethyltransferase [EC 2.1.2.1] were purified to homogeneity from a whole homogenate of rat liver without the prior separation of mitochondria. The molecular weight of the cytosolic enzyme was 230,000, and that of the mitochondrial enzyme was 200,000. Each of the isozymes contained 4 mol of pyridoxal 5'-phosphate/mol. Tryptic peptide analyses of the NaBH4-reduced and carboxymethylated isozymes showed that each contained a single peptide containing phosphopyridoxyllysine. The numbers of peptides obtained were about one-fourth of those expected from their contents of lysine plus arginine residues. These findings together with the identity of subunit molecular weight indicate that each of the isozymes is composed of 4 identical polypeptide chains. The isoelectric pH values of the cytosolic and mitochondrial enzymes were 4.95 and 5.30, respectively. Other differences between the isozymes include the amino acid composition, stability of the apoenzyme, reactivity toward L-allothreonine, and immunochemical properties.