This study investigated the subcellular distribution of Cu, Zn, Cd and Ag in liver of mallard (Anas platyrhynchos), spot-billed duck (A. poecilorhyncha) and great cormorant (Phalacrocorax carbo) to better understand metal fractionation and dynamics in avian liver. Most of the total Cu, Zn, Cd and Ag were present in hepatocytosol, and their concentrations increased with total hepatic levels in all the three avian species. Copper, Zn and Cd in hepatocytosol were present mostly in metallothionein fractions (MTs), and Cu and Cd contents in this fraction were positively correlated with hepatocytosol levels in all the species. Silver was observed mostly in both high-molecular weight and MT fractions in hepatocytosol in mallard and great cormorant, whereas it was present in both low-molecular weight and MT fractions in spot-billed duck, suggesting that distribution of Ag in the hepatocytosol was species-specific. The elution profile of metals in MT fractions revealed six metallic peaks in mallard and spot-billed duck, and three peaks in great cormorant, implying the presence of multiple MT isoforms in the liver of these avian species. The present study demonstrated that MTs are closely associated with metal regulation, especially Cu and Cd, in these three aquatic birds.