The methyl carbon of ribothymidine in Loop IV of the tRNA of Streptococcus faecalis, Bacillus subtilis, and some other microorganisms is derived directly from 5,10-methylenetetrahydrofolate, not S-adenosylmethionine. The pure enzyme from S. faecalis also requires FADH2. We have obtained evidence that tetrahydrofolate is a product of the reaction and demonstrated that label from [5-3H]5-deazaFMNH2 is incorporated into the methyl moiety of ribothymidine. These data indicate that the enzyme uses methylenetetrahydrofolate solely as a 1-carbon donor and employs FADH2 as a reducing agent in vitro according to the following reaction: tRNA(U psi C) + CH2 = THF + FADH2 leads to tRNA(T psi C) + THF + FAD.