The insect cell line derived from Spodoptera frugiperda (Sf9) does not express the activities of the trifunctional NADP-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase. The lack of synthetase activity was confirmed by the inability to incorporate radiolabeled formate into nucleotides. The cells express, instead, a Mg2+ and NAD-dependent bifunctional methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase with properties similar to the enzyme found in the mitochondria of transformed mammalian cells. In contrast, the enzyme in Sf9 cells is localized in the cytoplasm. Nutritional studies in defined medium with dialyzed serum demonstrated that the Sf9 cell does not required added purines or pyrimidines for growth. It is auxotrophic for cysteine and glycine; this latter requirement is probably due to the absence of mitochondrial serine hydroxymethyltransferase. Incorporation of labeled glycine and serine into DNA indicates that only serine is a source of one-carbon units. These results suggest that the mitochondria in Sf9 cells do not play a major role in folate-mediated metabolism.