Metallothionein (MT) has been used in immunohistochemical techniques to indicate presence and distribution of heavy metals within biological tissues. This study describes a comparison of the pattern of MT-immunostaining in the liver, duodenum and kidney during dietary copper overload in rats. Sixteen male 10-week-old Wistar rats were randomly allocated into groups of four. Two groups were fed a pelleted diet containing 1,500 mg/kg copper and two control groups received a rodent diet containing 10 mg/kg copper. After 6 weeks samples of liver, kidney and duodenum were collected for immunohistochemistry and histology. An indirect immunoperoxidase technique, using monoclonal antibody E9 against horse MT and polyclonal sera against rabbit MT, was employed. Copper-loaded rats had marked MT-immunoreactivity within the nucleus and cytoplasm of many periportal hepatocytes, renal proximal convoluted tubule epithelial cells, intestinal columnar epithelial cells and Paneth cells. Immunohistochemical staining was similar using either mouse anti-MT polyclonal serum, or monoclonal antibody E9. Hepatocytes surrounding inflammatory foci were positive for MT, supporting the proposed role of this protein in free radical scavenging. The presence of MT in the kidney appears to be associated with renal excretion of copper-metallothionein (Cu-MT) in copper-loaded rats. Paneth cells were easily detected using MT-immunostaining. MT may play a part in absorption of copper from intestinal contents and possible storage as Cu-MT in Paneth cells. The function of Paneth cells remains unknown but the presence of marked MT-immunoreactivity in these cells, observed in copper-loaded rats, suggests their involvement in homeostasis and metabolism of copper.