Objective To determine whether glyoxal can be converted to oxalate in human erythrocytes. Glyoxal synthesis is elevated in diabetes, cardiovascular disease, and other diseases with significant oxidative stress. Erythrocytes are a good model system for such studies as they lack intracellular organelles and have a simplified metabolism. Materials and Methods Erythrocytes were isolated from healthy volunteers and incubated with varying concentrations of glyoxal for different amounts of time. Metabolic inhibitors were used to help characterize metabolic steps. The conversion of glyoxal to glycolate and oxalate in the incubation medium was determined by chromatographic techniques. Results The bulk of the glyoxal was converted to glycolate, but ∼1% was converted to oxalate. Inclusion of the pro-oxidant, menadione, in the medium increased oxalate synthesis, and the inclusion of disulfiram, an inhibitor of aldehyde dehydrogenase activity, decreased oxalate synthesis. Conclusion The glyoxalase system, which utilizes glutathione as a cofactor, converts the majority of the glyoxal taken up by erythrocytes to glycolate, but a small portion is converted to oxalate. A reduction in intracellular glutathione increases oxalate synthesis and a decrease in aldehyde dehydrogenase activity lowers oxalate synthesis and suggests that glyoxylate is an intermediate. Thus, oxidative stress in tissues could potentially increase oxalate synthesis.