Although expression of the enzyme gamma-glutamyl transpeptidase (GGT) is the most common phenotypic marker of preneoplastic foci in the livers of carcinogen-treated rats, it is not generally expressed in mouse liver tumors or hepatic foci. However, several carcinogens, including safrole and ortho-azoaminotoluene (OAT), have been reported to induce GGT-positive foci in mice. We asked whether safrole and OAT induce GGT expression in preneoplastic foci or if these compounds select for a distinct set of lesions that can be identified by their GGT-positive phenotype. We treated 12-day-old male and female C57BL/6J mice with N,N-diethylnitrosamine (DEN) (0.20 mumol/g body wt) to initiate hepatocarcinogenesis. From 6 to 24 weeks of age, during the promotion phase of hepatocarcinogenesis, groups of mice were treated with 3,4,5,3',4',5'-hexabromobiphenyl (HBB), safrole or OAT. Additional groups of female mice were ovariectomized at 6 weeks of age with or without subsequent chronic treatment with testosterone. All the animals were killed at 24 weeks of age and serial liver sections were stained for glucose-6-phosphatase (G6Pase) or GGT. Both testosterone and HBB were strong promoters of the development of G6Pase-deficient foci. No GGT-positive foci were observed in animals treated with these agents or with DEN alone. In mice fed safrole or OAT during the promotion period, female mice developed more G6Pase-deficient foci than male mice, and GGT-positive foci were observed. Analysis of serial sections revealed that the G6Pase-deficient foci and the GGT-positive foci were independent populations. The relative number of these two classes of foci varied according to the treatment regimen. In females fed safrole, 7% of the foci in the liver were GGT-positive while in female mice fed OAT, 45% were GGT-positive. In all groups of mice in which we observed GGT-positive foci and in ovariectomized female mice, we noted a third independent population of foci which demonstrated significantly increased expression of G6Pase relative to surrounding normal liver. These data indicate that different treatments during the promotion stage of hepatocarcinogenesis in the mouse may give rise to distinct populations of preneoplastic lesions. Further studies of the molecular events giving rise to these distinct lesions will provide insights into the multiple pathways that result in hepatocarcinogenesis.