Freshwater Oreochromis mossambicus (tilapia) were exposed to 3.2 µmol/l Cu(NO₃)₂ in the water for up to 80 days, and copper (Cu) and immunoreactive metallothionein (irMT) were localized in the branchial epithelium. Cu was demonstrated in mucous cells (MC), chloride cells (CC), pavement cells (PC), respiratory cells (RC), and basal layer cells (BLC) via autometallography combined with alcian blue staining for MC and Na⁺-K⁺-ATPase immunostaining for CC and, on the basis of their location in the epithelium of PC, RC, and BLC. In control fish (water with Cu concentration 90 nmol/l) incidentally irMT was observed in the area where progenitor cells of the branchial epithelia reside, as demonstrated by proliferating cell nuclear antigen staining. This was also the area where the first increase irMT expression of the Cu exposure was observed. After 2 days of exposure to Cu, irMT was found in CC and PC. From 5 days on, a pronounced irMT staining was observed in BLC of branchial epithelium, which then appeared to migrate and differentiate into mature CC, PC, and RC. We conclude that MT expression in mature CC, PC, and RC requires exposure to Cu in a earlier stage of development of these cells. Once expression is initiated in undifferentiated cells, MT remains expressed throughout the life cycle of the cell.