We have described new well-differentiated mouse hepatocyte-like cell lines (mhAT) derived from transgenic mice expressing simian virus 40 large T antigen under the control of antithrombin III gene promoter (Exp. Cell Res. (1992) 200, 175-185). In an attempt to understand the phenotypic variations of the different cell lines, we analyzed their content in liver-specific transcription factors at the level of both the proteins, by gel shift analysis, and the mRNA, by quantitative reverse-transcription PCR. Moreover, the transactivating ability of endogenous HNF1 alpha and C/EBP alpha was also evaluated by measuring the activity of transfected synthetic promoters consisting of DNA element homopolymers upstream of a TATA box. High levels of HNF1, HNF3, and HNF4 transcription factors were maintained in mhAT cells. In contrast, C/EBP alpha was much more variable between the different cell lines and was less abundant than it was in vivo, in the liver. We investigated the influence of HNF1 alpha and C/EBP alpha on the activity of transfected liver-specific promoters. HNF1 alpha was not limiting for the activity of transfected liver-type pyruvate kinase and albumin promoters. In contrast, the activity of the albumin promoter in the different lines was clearly dependent on the C/EBP alpha content, which seems, therefore, to be an essential factor modulating the expression of this gene in HNF1 alpha-containing cells. This work shows that the correlations between promoter activities and transacting factor contents in a panel of well-differentiated cultured cells can be used to determine the respective role of transcription factors on the strength of some promoters.