We optimally solubilized the diformazan that forms during the fructosamine reaction with a nonionic detergent; we then automated the assay. After optimal solubilization of diformazan, we found that serum urate contributed significantly to the color formation in fructosamine reaction. When the error introduced by urate was corrected mathematically by use of an experimental determined factor, the serum fructosamine concentrations obtained were comparable with those of a more specific test of the nonenzymatic glycation (HPLC of hydrolyzed proteins). We found that concentrations of fructosamine in serum increased with increasing age of subjects. In diabetic patients the average concentration of fructosamine in serum exceeded that in nondiabetic subjects and correlated with the preprandial serum glucose and with other measures of deteriorating glycemic control (e.g., increased need for insulin therapy). Serum fructosamine normalized for albumin content was also increased in patients with chronic renal failure. Multiple mechanisms (including exposure of patients to hypertonic glucose during dialysis) appear to be involved in the increase of serum fructosamine in patients with chronic renal failure.