Carcinogenesis develops in stages that have been operationally defined as initiation, promotion and progression. Although morphological end points have been described for detection and quantitation of these stages, to date initiation has been assessed only in the context of clonal growth in response to certain promoting agents. Initiated cells are morphologically indistinguishable from surrounding cells and early changes at the cellular level during initiation have not been clarified. One commonly used end point for the detection of preneoplastic hepatic lesions i their aberrant expression of the placental isozyme of glutathione S-transferase (PGST). Because single hepatocytes expressing PGST have been detected in aged rats and in those administered hepatocarcinogens, it has been suggested that such cells constitute a population of putatively initiated hepatocytes. In order to further elucidate the characteristics of single PGST-positive hepatocytes, we analyzed the number of these cells 2 and 18 weeks after various doses (0-100 mg/kg) of diethylnitrosamine (DEN) and of dimethylbenz[a]anthracene (DMBA). When determined 14 days after carcinogen administration, the number of single hepatocytes expressing PGST was greater after DEN administration (ranging from 0.8 +/- 0.3 per cm2 transection of liver at 1 mg/kg to 33.0 +/- 4.7 at 100 mg/kg) than after DMBA administration (ranging from 0.25 +/- 0.14 at 10 mg/kg to 3.03 +/- 0.5 at 100 mg/kg); none were detected in control rats of the same age. Additional rats were maintained on a basal diet or a basal diet plus phenobarbital for a further 4 month period. Whereas individual PGST-positive hepatocytes were only sporadically detected in rats treated with DMBA and maintained on a basal diet for 18 weeks, those rats placed on phenobarbital for 16 weeks had an even higher number of such PGST-positive hepatocytes than at 2 weeks after DMBA administration. In contrast, the dose-response curve observed for DEN-treated rats 18 weeks after carcinogen administration was similar to that observed 2 weeks after carcinogen treatment for both phenobarbital- and non-phenobarbital-treated rats. In addition, the number of single PGST-positive hepatocytes detected at 2 weeks was directly parallel to the number of altered hepatic foci expressing PGST 18 weeks after DEN administration. The dose-dependent induction of PGST-positive single hepatocytes after treatment with two hepatocarcinogens, the dose-dependent growth of altered hepatic foci (AHF) expressing PGST with phenobarbital administration and the parallel dose-response curve of single hepatocytes expressing PGST and later of AHF expressing PGST argue strongly for a precursor role of single PGST-positive cells in the development of AHF expressing PGST.