2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent tumor promoter in two-stage models of hepatocarcinogenesis. This study focuses on the persistence or reversibility of TCDD-mediated changes in livers after 30 weeks of treatment and cessation of treatment. Diethylnitrosamine (DEN) initiated animals (175 mg/kg) were promoted bi-weekly with TCDD at a dose equivalent to 125 ng/kg/day for 30 weeks without or with a following waiting period of 32 weeks before necropsy. 2,3,7,8-Tetrachlorodibenzo-p-dioxin liver concentration decreased 300-fold above background. Induction of CYP1A1 dependent enzyme activity decreased according to TCDD tissue levels. In contrast, cell proliferation, as measured by BrdU-labeling index, was still 2.8-fold increased over controls in the TCDD group with waiting period compared to a 4-fold increase over controls at the end of the 30 week dosing period. Enzyme altered hepatic foci expressing the placental form of glutathione S-transferase decreased in number but the remaining foci were significantly increased in size and the percent of liver occupied by foci was higher at the end of the waiting period as compared to livers at the end of the dosing period. Liver tumor incidence at the end of the waiting period was 71% (5 of 7 animals) and the livers showed an increase in bile duct lesions with only mild toxicity. There was pronounced bile duct proliferation in DEN/TCDD treated animals after the waiting period with intense expression of TGF alpha in bile duct epithelial cells at detected by immunohistochemical methods. In comparison, at the end of the 30 week dosing period the livers showed more severe toxicity and only mild bile duct proliferation. Also, one small hepatocellular adenoma was observed. It is concluded that as opposed to CYP1A1 induction the more complex biological responses, cell proliferation and selective growth of certain preneoplastic foci, are persistent after prolonged TCDD treatment within the experimental framework of our study.