Cholangiocarcinoma (CCA) is a lethal disease, afflicting many thousands the world over. Human CCA develops through a multi-step progression model, preceded by the onset of dysplasia in the cholangiolar ductal epithelium. An animal model of multi-step carcinogenesis in the biliary tree will enable the study of genetic changes in human CCA, and provide an avenue for chemoprevention strategies. We describe an oral thioacetamide (TAA)-induced model of rat CCA that recapitulates the histologic progression of human CCA. Male Sprague-Dawley (SD) rats (n = 170), weighing 350 +/- 20 g, were used in this study. Drinking water with TAA 300 mg/l was administered orally, and the liver was harvested and examined histologically at weekly intervals, beginning at 5 weeks after initiation of TAA. Harvested tissues were formalin-fixed and paraffin embedded for morphologic and immunohistochemical studies. Multifocal bile ductular proliferation with intestinal metaplasia (presence of goblet cells) and increasing histologic atypia (biliary dysplasia) was observed by the 9th week of TAA administration. Biliary cytokeratin (CK19)-expressing invasive intestinal-type CCA with stromal desmoplasia was evident at the 16th week, and by the 22nd week, the yield rate for CCAs had increased to 100%. Invasive CCAs preceded the development of hepatic cirrhosis by at least 4 weeks; the earliest incidence of hepatic fibrosis was observed beginning at 20 weeks post-TAA administration. The progression from normal cholangioles to biliary dysplasia to invasive CCA was accompanied by up-regulation of the proto-oncogenes c-met and c-erbB-2, tyrosine kinase receptors over-expressed in human CCAs. The study was terminated at 6 months, at which time no systemic metastases or deaths were observed. Oral administration of TAA in drinking water to male SD rats provides a reproducible animal model for development of CCA with a high yield rate. In particular, the presence of biliary dysplasia beginning at the 9th week, which progresses to invasive CCA, mimics the multi-step model of human CCA. The TAA rat model may serve as a powerful pre-clinical platform for therapeutic and chemoprevention strategies for human CCA.