Only two S-adenosyl-l-methionine synthetase forms exist in rat liver: high-MrS-adenosyl-l-methionine synthetase and low-MrS-adenosyl-l-methionine synthetase, which have been purified to apparent homogeneity as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. High-MrS-adenosyl-l-methionine synthetase had an apparent molecular mass, determined by gel filtration, of 210 kDa and was a tetramer constituted by 48.5-kDa subunits, estimated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The apparent molecular mass of low-MrS-adenosyl-l-methionine synthetase, as estimated by gel filtration, was 110 kDa and was constituted by two subunits of 47 kDa. An antiserum against low-MrS-adenosyl-l-methionine synthetase cross-reacted with the two forms. Reverse-phase HPLC runs of tryptic digestions of high-Mr and low-MrS-adenosyl-l-methionine synthetase showed that the peptide maps of the two forms were very similar, if not identical. High-MrS-adenosyl-l-methionine synthetase activity was inhibited by S-adenosyl-l-methionine and pyrophosphate. Depending on the dose used, S-adenosyl-l-methionine activated or inhibited low-MrS-adenosyl-l-methionine synthetase and pyrophosphate had no effect on this form. The two synthetases showed a different specific activity at the physiological concentration of methionine. This report shows that even though the two forms are constructed of the same polypeptide chains, they are regulated in a different manner by methionine and by the products of the reaction.