The human hepatoma HepaRG cells are able to differentiate in vitro into hepatocyte-like cells and to express various liver-specific functions, including the major cytochromes P450. This study was aimed to determine whether differentiated HepaRG cells retained their specific functional capacities for a long time period at confluence. We show that expression of transcripts encoding CYP1A2, 2B6, 3A4, and 2E1, several phase II and antioxidant enzymes, membrane transporters, including organic cation transporter 1 and bile salt export pump, the nuclear receptors constitutive androstane receptor and pregnane X receptor, and aldolase B remained relatively stable for at least the 4-week confluence period tested. Similarly, activities of CYP3A4 and CYP1A2 and their responsiveness to prototypical inducers were well preserved. Aflatoxin B(1), a potent hepatotoxicant and carcinogen, induced a dose-dependent and cumulative cytotoxicity. Furthermore, at a concentration as low as 0.1 microM, this mycotoxin caused a decrease in both CYP3A4 activity and intracellular ATP associated with morphological alterations, after 14 days following every 2-day exposure. Moreover, using the comet assay, a dose-dependent DNA damage was observed after a 3-h treatment of differentiated HepaRG cells with 1 to 5 microM aflatoxin B(1) in the absence of any cell damage, and this DNA damaging effect was strongly reduced in the presence of ketoconazole, a CYP3A4 inhibitor. These results bring the first demonstration of long-term stable expression of liver-specific markers in HepaRG hepatocyte cultures maintained at confluence and show that these cells represent a suitable in vitro liver cell model for analysis of acute and chronic toxicity as well as genotoxicity of chemicals in human liver.