A panel of four novel human hepatoma cell lines was isolated from a single tumor from a male individual. BC1, B16 and B16A2 lines were well differentiated, while cells of the B9 line were only poorly differentiated, being essentially negative for the functions analyzed. These cell lines have been surveyed for expression of a large set of plasma proteins, accumulation of liver-specific mRNAs and DNA-binding activity of ubiquitous and liver-enriched transcription factors. BC1 cells expressed the highest levels of albumin mRNA, whereas B16 and B16A2 cells accumulated the largest amounts of haptoglobin mRNA. In addition, B16 and B16A2 cells were unique in that they expressed CYP2E1 mRNA, a species absent from the available human liver cells, including HepG2 hepatoma cells, and 3-methylcholanthrene-inducible CYP1A2 mRNA. The activities of genes encoding transcription factors were evidenced in all four cell lines which expressed mRNAs for nuclear factor interleukin 6 and hepatocyte nuclear factor 1 (HNF) together with the DNA-binding activity of NFY and AP1 nuclear proteins. Strikingly, HNF-1 and HNF-4-like DNA-binding activities were restricted to BC1, B16 and B16A2 cells, supporting the idea of the potential role of these (or closely related) factors in the maintenance and/or in the establishment of the differentiated phenotype. B9 cells contained variant HNF1-like DNA-binding activity, similar to dedifferentiated rat hepatoma cells of the H5 line. CCAAT/enhancer-binding protein and HNF-3-like activities were found in all cell lines, although at a lower level and/or activity in B9 cells. Finally, transfection experiments of plasmids containing the whole hepatitis-B virus genome demonstrated that B16 cells, but not B9 cells, were able to support hepatitis-B virus replication and virion production, in agreement with the notion that HNF-1 activity is necessary for viral replication. We believe that the specific complement of transcription factors expressed in the differentiated BC1, B16 and B16A2 cells, and in the poorly differentiated B9 cells, will allow studies on the regulation of hepatic gene expression in these human lines, and will also aid the analysis of xenobiotic metabolism and the biology of hepatitis-B virus replication.