Three isozymes of methionine adenosyltransferase (EC 2.5.1.6; MAT-I, -II, and -III) exist in normal rat liver and are conveniently purified (MAT-III to homogeneity) by a three-step column chromatography procedure. MAT-I shows Michaelis-Menten kinetics with a Km (L-methionine) of 41 microM and a molecular weight of 208 000 and is slightly inhibited by S-adenosyl-L-methionine (Adomet). MAT-II, which is also the only isozyme found in normal rat kidney, shows negative cooperativity with a Hill coefficient of 0.7. It has a L-methionine concentration required for half-maximal velocity [S0.5(Met)] of 8 microM and a molecular weight of 120 000 and is strongly inhibited by Adomet. MAT-I and -II comprise 15% and 5%, respectively, of total MAT activity in rat liver. The predominant isozyme in rat liver, MAT-III, demonstrates positive cooperativity with a Hill coefficient of 1.8. It has a molecular weight of 97 000 and apparently consists of two subunits of identical molecular weight (47 000). This liver-specific isozyme is strongly activated by both dimethyl sulfoxide and Adomet and has a S0.5(Met) of 215 microM.