Abstract: The steric course of the conversion of glycine to 5, lO-methylenetetrahydrofolate (5, lO-CH2-H4folate) by the glycine cleavage system in E. coli has been elucidated. 2-H, e of glycine is the precursor of 11-H, of 5, lO-CH2-H, folate, and 2-Hsi of glycine is the precursor of 1 l-Hsi of 5, lO-CH2-H4folate. The overall transformation is at least 78% stereospecific (possibly higher).