The level of DNA fragmentation, as evaluated by alkaline elution, and of unscheduled DNA synthesis (UDS), as measured by autoradiography, was determined in the parenchymal cells from the entire liver during the development of hyperplastic lesions induced in the rat by the following treatment: diethylnitrosamine (DEN) (200 mg/kg i.p.) on Day 0; CCl4 (2 ml/kg intragastrically) on Day 21; dietary administration of 0.02% 2-acetylaminofluorene during the third and the fourth wk; and of 0.05% phenobarbital from the sixth wk. Both DNA fragmentation and UDS were constantly detected, concomitantly with the presence of gamma-glutamyltransferase (gamma-GT)-positive hepatocytes, in the primary cultures derived from the liver of rats of this experimental group sacrificed at 4, 5, 6, and 7 wk after DEN injection, their amount being approximately the same at the fourth and at seventh wk. Moreover, evidence of DNA alterations was still present, albeit diminished, 22 wk after the beginning of treatment. In contrast, DNA fragmentation and UDS did not persist past the fifth wk, and gamma-GT-positive hepatocytes were very few or totally absent in hepatocyte primary cultures from control rats treated with DEN alone, 2-acetylaminofluorene alone, or 2-acetylaminofluorene:CCl4. CCl4 alone, and phenobarbital alone caused only a modest, albeit statistically significant, increase in DNA elution rate and UDS, respectively. In a comparison performed on hepatocyte primary cultures obtained from rats of the experimental group sacrificed at the fifth wk after the injection of DEN, the level of UDS was higher in gamma-GT-positive than in gamma-GT-negative hepatocytes. These results indicate that the regimen used to induce the selective proliferation of initiated hepatocytes actually produces extensive DNA lesions which can give rise to additional carcinogenic initiations.