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Metallothioneins (MTs) are low molecular weight ubiquitous metalloproteins with high cysteine (thiol) content. The intracellular concentration of zinc (Zn) is tightly regulated and MT plays a crucial role in it. The present study investigates the relationship between the Zn status (as a function of Zn concentration and time) in the rat liver and the occurrence of hepatic MT. For dose dependent study, four experimental groups, one control and three receiving different levels of metal supplementation, were chosen [Group 1 control and Group 2, Group 3, Group 4 receiving subcutaneous dose of 10, 50 and 100 mg of Zn/kg body weight (in the form of ZnSO·7HO), respectively]. For the time dependent expression of MT, again four experimental groups, i.e. Group 5 control and Group 6, Group 7, Group 8 receiving 50 mg of Zn/kg body weight (in the form of ZnSO·7HO) subcutaneously and sacrificed at different time intervals after last injection i.e. 6, 18, 48 h, respectively were chosen. Isolation of MT was done by using combination of gel filtration and ion exchange chromatography while characterization of MT fraction was carried in the wavelength range 200-400 nm. Expression of MT was studied by using Western blot analysis. The results revealed that the MT expression increases with increasing the dose of Zn administered and maximum at 18 h after last Zn injection. Accumulation of MT with increase dose would help in maintaining the intracellular Zn concentration by its sequestration which further reduces the possibility of undesirable binding of Zn to other proteins significantly and maintains Zn homeostasis. The maximum expression of MT at 18 h is indicative of its half life.