The carcinogenic potential of phenobarbital (PB) and 3-methylcholanthrene (3-MC) was assayed in a new medium-term carcinogenicity bioassay using D-galactosamine (DCA) as a nonsurgical method to induce liver cell regeneration in place of partial hepatectomy (PH). Rats were initially given a single ip injection (200 mg/kg) of diethylnitrosamine (DEN) and after 2 wk on basal diet received 2 ip injections of DCA (300 mg/kg) at the end of wk 2 and 5. They were treated with one of the test compounds PB or 3-MC in the diet or fed basal diet for wk 3-8. Carcinogenic potential was assessed by comparing the numbers and areas per square centimeter of glutathione S-transferase placental form-positive (CST-P*) foci in the livers of test chemical-treated animals with those of the control animals given DEN/DCA alone. Positive estimations of carcinogenicity were obtained for PB, which is a nongenotoxic liver tumor promoter, and for 3-MC, which is a genotoxic nonliver carcinogen. Increases of liver/body weight ratios and serum total cholesterol were observed in rats treated with PB or 3-MC. Interestingly, interlobe differences were found on the development of CST-P* liver cell foci. Our results thus confirm that the present bioassay protocol with repeated administration of DCA instead of PH may offer a new and sensitive method to screen large numbers of environmental liver and nonliver carcinogens.