Oxalate synthesis in human hepatocytes is not well defined despite the clinical significance of its overproduction in diseases such as the primary hyperoxalurias. To further define these steps, the metabolism to oxalate of the oxalate precursors, glycolate and glyoxylate, and the possible pathways involved were examined in Hep G2 cells. These cells were found to contain oxalate, glyoxylate and glycolate as intracellular metabolites, and excrete oxalate and glycolate into the medium. Glycolate was taken up more effectively by cells than glyoxylate, but glyoxylate was more efficiently converted to oxalate. Oxalate was formed from exogenous glycolate only when cells were exposed to high concentrations. Peroxisomes in Hep G2 cells, in contrast to those in human hepatocytes, were not involved in glycolate metabolism. Incubations with purified lactate dehydrogenase suggested that this enzyme was responsible for the metabolism of glycolate to oxalate in Hep G2 cells. The formation of ¹⁴C-glycine from ¹⁴C-glycolate was observed only when cell membranes were permeabilized with Triton X-100. These results imply that peroxisome permeability to glycolate is restricted in these cells. Mitochondria, which produce glyoxylate from hydroxyproline metabolism, contained both alanine:glyoxylate aminotransferase type 2 (AGT2) and glyoxylate reductase activities, which can convert glyoxylate to glycine and glycolate, respectively. Expression of AGT2 mRNA in Hep G2 cells was confirmed by RT-PCR. These results indicate that Hep G2 cells will be useful in clarifying the non-peroxisomal metabolism associated with oxalate synthesis in human hepatocytes.