Complementation analysis at the single gene level has been carried out on 13 different, recessive, glycine-requiring mutants produced by action of mutagenic agents on the CHO-K1 Chinese hamster cell. The mutants form four different complementation classes indicating that at least four separate genes can produce a growth requirement for glycine. The four classes of mutants can be distinguished biochemically as well as genetically. Existence of these four mutant classes appears to explain the preponderance of the glycine-requiring forms in the auxotrophs produced by treatment of the parental cell with mutagenic agents.