The folate-binding protein of rat liver mitochondria [Zamierowski, M. & Wagner, C. (1977) J. Biol. Chem. 252, 933-938] has been purified to homogeneity by a combination of gel filtration, DEAE-cellulose, and affinity chromatography. This protein was assayed by its ability to bind tetrahydro[3H]folic acid in vitro. the purified protein contains tightly bound flavin and has a molecular weight of about 90,000 as determined by sodium dodecyl sulfate electrophoresis. This protein also displays dimethylglycine dehydrogenase [N,N-dimethylglycine: (acceptor) oxidoreductase (demethylating), EC 184.108.40.206] activity which copurifies with the folate-binding activity. It is suggested that the role of the tetrahydrofolic acid is to accept the formaldehyde produced during the course of the reaction.