The pathways of oxalate synthesis in humans are not well defined despite their clinical significance in primary hyperoxaluria and idiopathic calcium oxalate nephrolithiasis. Furthermore, the functional roles, if any, of this synthesis have not been elucidated. This study examines pathways of oxalate synthesis from glycolate in Hep G2 cells, a human hepatoma cell line. Incubation of these cells with glycolate has revealed that a pathway may function to synthesize oxalate from glycolate that does not depend on the oxidation of glycolate to glyoxylate by glycolate oxidase. Labeling cells with 14C-glycolate and chromatographic analyses indicated that detectable amounts of 14C-glyoxylate were not formed. A radioactive peak that coeluted with oxalate on ion exclusion chromatography was the only peak yet identified. A detailed examination of glycolate metabolism in these cells should help clarify the terminal steps associated with oxalate synthesis and aid in our understanding of two-carbon metabolism.