A pathway resulting in the formation of methionine from the 5'-methylthioadenosine is shown in vitro using cell-free homogenates of rat liver. Under the conditions used, methionine was the major product produced, as determined by its chemical and chromatographic properties. The kinetics of product formation indicated that 5'-methylthioadenosine is first rapidly converted to 5-methylthioribose 1-phosphate, followed by its slower conversion to methionine. 5'-[Methyl-14C]methylthioadenosine, 5'-[methyl-3H]-methylthioadenosine, 5'-[35S]methylthioadenosine, and 5'-[adenosine-U-14C]methylthioadenosine were synthesized to determine which portion of the molecule became incorporated into methionine. Carbons from the ribose portion, the carbon and hydrogens of the methyl group, and the sulfur of 5'-methylthioadenosine are all incorporated into methionine. The ratio of incorporation of the sulfur and the methyl carbon was 1:1. Therefore, the pathway for methionine synthesis involves modifying the ribose portion of the 5'-methylthioadenosine into the 2-aminobutyrate portion of methionine, with the thiomethyl group remaining intact. This pathway appears to be a significant salvage pathway for methionine synthesis in mammals, and may be necessary for removal of 5'-methylthioadenosine produced as a by-product of polyamine biosynthesis.