Metallothionein (MT) in rat liver was separated and determined by high-performance liquid chromatography (HPLC) with a Superdex 75 high-performance gel filtration column. For sample preparation, the rat liver was homogenized in saline and the homogenate was centrifuged at 100,000xg for 60 min. The supernatant was saturated with cadmium solution, heated and centrifuged before HPLC determination. The mobile phase for HPLC was 0.1M phosphate-0.2M NaCl (pH 7.4) at a flow rate of 1.0ml/min. The MT was detected by a UV spectrophotometric detector at 250nm. The retention time of MT was 14.3min. The coefficient of variation of the method was 1.1% and 1.4% for the standard and sample, respectively. The recovery was 91.5%-92.4% for different levels of MT standard addition. The detection limit for MT was 0.265 micrograms, and it was possible to detect an MT content of about 13 micrograms/g wet wt in rat liver. Between the MT values determined by the traditional cadmium saturation method and those by this method, a good correlation (r = 0.99) was found. The method reported is relatively simple for determination of MT in rat liver and can determine metalloprotein content directly.