Cultured Chinese hamster ovary cells accumulated labeled folate and 5-methyltetrahydrofolate and metabolized these compounds to polyglutamates of chain length up to 10, with hexa-, hepta-, and octaglutamates predominating. Intracellular vitamin was concentrated up to 50-fold, although the intracellular pteroylmonoglutamate concentration did not exceed that in the medium. Folylpolyglutamate levels in mutant cells (AUXB1), which are defective in the enzyme folylpolyglutamate synthetase, were less than 1.5% of wild type levels, while pteroylmonoglutamate levels were normal. Tetrahydropteroylpolyglutamates were the most effective polyglutamate substrates for the Chinese hamster ovary cell folylpolyglutamate synthetase. The final distribution of folylpolyglutamates in this cells appeared to be a reflection of the ability of different glutamate chain length folates to act as substrates for the synthetase. The proportions of individual folylpolyglutamates were unaffected by culturing the cells in medium containing or lacking products of one carbon metabolism such as glycine, adenosine, or thymidine. However, these proportions were markedly influenced by the level of methionine in the culture medium. The possible significance of changes in the glutamate chain length of folates under different nutritional conditions in relationship to the regulation of one carbon metabolism is discussed.