An initiation promotion bioassay for chemical carcinogens and tumor promoters has been developed in rat liver using presumed preneoplastic lesions, foci of gamma-glutamyltranspeptidase (GGTase)-positive hepatocytes, as the endpoint. To evaluate the tumor-promoting activity of phenobarbital, rats were administered diethylnitrosamine (DENA), 2.0 mmole/kg, followed by 500 ppm phenobarbital in their drinking water. After 6 weeks of phenobarbital promotion, the rats, had an increased incidence of foci as compared to nonphenobarbital-treated rats. By 50 weeks, the number of foci in the nonpromoted animals equaled the number observed with promotion. The stability and progression of GGTase-positive foci was determined in rats that received a 2/3 partial hepatectomy, followed 24 hours later by DENA administration (0.3 mmole/kg). The rats then received 500 ppm phenobarbital in the drinking water for 7 weeks. After 7 weeks, half of the rats were continued on phenobarbital and the other half were removed from phenobarbital treatment.