Immunohistochemical localization of gamma-glutamyl transpeptidase (gamma-GTP) in rat liver during 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) hepatocarcinogenesis was investigated and compared with sites of gamma-GTP activity. Immunohistochemically, gamma-GTP was stained in the apical border of proliferating oval cells during the early stages of azo-dye carcinogen feeding. After 7 weeks, multiple hyperplastic nodules appeared in which gamma-GTP was localized in the bile canaliculi. In hepatoma tissues, positive staining for gamma-GTP was observed in the bile canaliculi-like spaces, on the cell membrane, and sometimes in the cytoplasm of malignant cells. Enzyme histochemical staining showed gamma-GTP activity to be present in almost the same areas as the immunoreactive gamma-GTP. However, some areas adjacent to hepatoma tissue showed immunohistochemically reactive protein but no enzyme activity. Immunoreactive gamma-GTP was present in all locations at which enzyme activity was seen. The present data suggest that an altered form of gamma-GTP might be present in tissues during 3'-Me-DAB hepatocarcinogenesis.