Hepatic oval cells (HOC) are considered to be the stem cells of the liver and have been linked to the development of hepatic malignancies. Studies have demonstrated that chronic hepatitis B virus (HBV) infection and dietary aflatoxin B-1 (AFB(1)) exposure are among the most important risk factors for the development of hepatocellular carcinoma (HCC). However, little research has been done to evaluate the role of oval cells in these two environmental factors on hepatocarcinogenesis. In this study, partial transformation of rat HOC (LE/6) were accomplished by transfected HBV x gene (HBx), and then transfected cells were implanted both intra-hepatically and subcutaneously into nude mice treated with AFB(1) in vivo. We found the oval cells produced tumors (4/24 of the animals) in liver following transfection with HBx gene and treatment with AFB(1). These intrahepatic tumors included HCC cells (immunopositive for HepParl, ALB, CK8 and AFP) and mesenchymal cells (immunopositive for Vimentin and SMA). Whereas mesenchymal tumors were observed at the subcutaneous tissue with a similar rate in all controls treated with cell lines (10/24 in HBx-oval cells/AFB(1) group, 8/20 in HBx-oval cells/non-AFB(1) group, 10/20 in non-HBx/AFB(1) group; 9/20 in non-HBx/non-AFB(1) group). Conversely, none of the controls developed intrahepatic tumors. These results provide an evidence that oval cells have the capacity to generate HCC through the combined effects of the HBx and AFB(1) in the liver microenvironnient. (C) 2011 Elsevier Ireland Ltd. All rights reserved.