In this study, we used [³H]thymidine labeling of newly synthesized DNA to examine the earliest effects of 2-acetylaminofluorene (2-AAF) on the mitotic activation of cells in the adult rat liver, and in situ hybridization analysis to study the expression of three transcription factors (HNF1β, HNF3γ, and HNF4), and two of the genes (α-fetoprotein [AFP] and albumin) regulated by these factors. A low dose of 2-AAF (and its analogs, 2-AF [2- aminofluorene] and N-OH-2-AAF) elicited a mitogenic response in ductal cells and nondescript periductular cells within 24 hours after administration. The compounds also induced the expression of HNF1β, HNF3γ, AFP, and albumin in ductal structures but had no detectable effect of HNF4 expression. In contrast, initiation of bile duct proliferation by ligation of the common bile duct had no effect on the expression of these genes in ductal cells. In addition to inducing a mitogenic response, 2-AAF resulted in increased numbers of apoptotic cells in the portal areas, a process that contributed to overall retention of liver morphology. Our results demonstrate that 2-AAF and some of its analogs can elicit a specific mitogenic response and induce expression of the 'establishment' transcription factors, HNF1β and HNF3γ, in ductal cells. Our data provide further support of a precursor-product relationship between 'stem-like' cells located in ductal structures, oval cells, and hepatocytes.