Previous studies reported that constitutive androstane receptor (CAR) does not transactivate phenobarbital responsive unit (PBRU)2C1luciferase reporter gene in COS cells in which endogenous CYP2B1 gene is not induced with PB. In order to understand molecular mechanism(s) whereby PBRU is transactivated, this article determined if the use of strong thymidine kinase (TK) promotor rather than the minimal CYP2C1 promotor, and hepatocyte nuclear factor-4 (HNF-4) can affect CAR-mediated transactivation of PBRU in the monkey kidney epithelial-derived COS-7 cells. To examine CAR-mediated transactivation, cultured COS-7 cells were transfected with CAR expression plasmid, pEGFP-mCAR1, and confirmed for high level of the protein expression. In COS-7 cells, TK promotor induced CAR-mediated PBRU transactivation in a dose-dependent manner. Whereas expression of HNF-4 slightly promoted PBRU transactivation with low amount of CAR transfected, it repressed PBRU transactivation in a dose-dependent manner with high amount of CAR. Consistent with the previous reports in Hep G2 cells, CAR transactivated PBRU2C1luciferase in a dose-dependent manner and this CAR-mediated transactivation required functional NR-1 and NF-1 sites. However, HNF-4 did not affect CAR-mediated PBRU transactivation in Hep G2 cells. These results suggest that proximal promotor and a trans-acting factor, HNF-4, can affect CAR-mediated transactivation of PBRU in COS-7 cells.