A histochemical procedure was used to determine the presence of gamma-glutamyl transpeptidase (GGT) in the livers of control, regenerating and carcinogen-treated mice. Young Balb/c mice were fed safrole, a naturally occurring hepatocarcinogen (0.4% w/w), for one year. Ten mice from control and ten mice from the safrole-treated group were killed at 4, 8, 16, 24, 36, and 52 weeks of exposure and 9 adult mice were killed after 2/3 hepatectomy. Basophilic and acidophilic foci of altered hepatocytes occurred in safrole-treated mice after 24 weeks. Neoplastic nodules appeared after 36 weeks. Both foci and nodules displayed elevated GGT activity as determined by enzyme histochemistry. Variability in the pattern of enzyme distribution and staining intensity was seen between cells of the same focus or nodule, as well as between different foci and nodules. Hepatocytes from regenerating livers of partially hepatectomized mice were negative for GGT. These results demonstrated a sequential development of carcinogen-altered hepatocyte populations characterized by the appearance of GGT activity prior to carcinoma formation. The results in the mouse show marked similarity to those reported in rat liver, where GGT has been used as a positive marker for premalignant liver lesions induced by a variety of carcinogens.