The estimation of metallothionein (MT) levels in a biological sample is required to study the physiological role or the induction of MT, which is a multifunctional protein. MT contains 61 amino acids with 20 homologous cysteines. The high selectivity, sensitivity and stability of N-[4-(dimethylamino-2-benzofuranyl)phenyl]maleimide (DBPM) for the determination of SH groups was very useful for the determination of MT. The detection limit of MT by this method was 10 ng/0.5 ml. This method made it possible to measure MT levels in normal tissues in combination with gel filtration chromatography. The age-dependent change in MT levels in normal rat tissues was also investigated by the present method. MT levels in normal rat tissues vary with age. It is thought that this fact is correlated with the biological roles of MT.