To clarify roles of intermediate metabolites of the glycolytic pathway and the polyol pathway in nonenzymatic glycation under physiological conditions, we incubated bovine serum albumin with intermediates of both pathways in the micromolar range as well as with 20 mmol/l glucose, and observed the formation of advanced glycation end products (AGEs). We found that triose phosphates, glyceraldehyde, and a novel polyol pathway-related metabolite, fructose 3-phosphate along with its breakdown product, 3-deoxyglucosone were extremely potent glycating agents that at nearly physiological concentrations on incubation with albumin produced substantial amounts of AGEs as early as 24 hours, while 20 mmol/l glucose afforded trace amounts of AGEs after two week incubation. The results along with the previous evidence of the increased level of intermediates in diabetic states may suggest that the intermediate metabolites rather than glucose contribute to enhanced glycation in diabetic tissues, inspite of the much lower concentrations compared with glucose.