A sensitive and specific method was developed for measuring medium-chain dicarboxylic acids (adipic and suberic acid) in urine. These acids were extracted from urine with diethyl ether and converted into fluorescent derivatives with 9-anthryldiazomethane, which can be separated by high-performance liquid chromatography. The reproducibility was high and the recovery from urine was above 90%. Urinary concentrations of adipic acid in streptozotocin-induced diabetic rats were significantly higher than those in control rats. In diabetic patients, both adipic acid and suberic acid tended to be high, but not significantly. This method should be useful for measuring dicarboxylic acids in urine.