The hepatic SV40 large T-antigen immortalized human liver epithelial (THLE) cell line and sublines transfected with cytochromes P450 (P450s) are increasingly being used for evaluation of potential drug-induced liver injury. So far, the available information on transporter and enzyme expression in these transfected cell systems is scattered. The purpose of this study was to characterize THLE cell lines with respect to transporter and enzyme expression. The mRNA expression of 96 typical drug absorption, distribution, metabolism and excretion genes, which encode a selection of transporters, phase I and II drug-metabolizing enzymes, and nuclear hormone receptors, was investigated in five THLE cell lines transfected with individual human P450s and in mock-transfected THLE-null cells using real-time polymerase chain reaction. The majority of the analyzed genes was either absent or expressed at low levels in the THLE-null and THLE-P450 cells, apart from housekeeping genes and the individual transfected P450s. Enzyme activity measurements provided confirmatory functional data for CYP2C9 and CYP3A4. Comparison with gene expression in human liver revealed an overall much lower gene expression in the THLE cell lines. The low levels of expression of a broad range of P450 genes in the THLE cell lines highlight the value of studies undertaken with P450-expressing cell lines for investigation of mechanisms of P450 metabolite-mediated hepatotoxicity. However, when attempting to translate between data obtained in THLE cell lines in vitro and functional consequences in vivo, it is important to take account of their limited expression of genes encoding many other drug-metabolizing enzymes and hepatic transporters.