Activating transcription factor (ATF) 5 is a member of the ATF/cAMP response element-binding protein family, which has been associated with differentiation, proliferation, and survival in several tissues and cell types. However, its role in the liver has not yet been investigated. We show herein that ATF5 is a highly abundant liver-enriched transcription factor (LETF) whose expression declines in correlation with the level of dedifferentiation in cultured human hepatocytes and cell lines. Re-expression of ATF5 in human HepG2 cells by adenoviral transduction resulted in a marked selective up-regulation of CYP2B6. Moreover, adenoviral cotransfection of ATF5 and constitutive androstane receptor (CAR) caused an additive increase in CYP2B6 mRNA. These results were confirmed in cultured human hepatocytes, where the cooperation of ATF5 and CAR not only increased CYP2B6 basal expression but also enhanced the induced levels after phenobarbital or 6-(4-chloropheny-l)-imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO). Comparative sequence analysis of ATF5 and ATF4, its closest homolog, showed a large conservation of the mRNA 5'-untranslated region organization, suggesting that ATF5 might be up-regulated by stress responses through a very similar translational mechanism. To investigate this possibility, we induced endoplasmic reticulum stress by means of amino acid limitation or selective chemicals, and assessed the time course response of ATF5 and CYP2B6. We found a post-transcriptional up-regulation of ATF5 and a parallel induction of CYP2B6 mRNA. Our findings uncover a new LETF coupled to the differentiated hepatic phenotype that cooperates with CAR in the regulation of drug-metabolizing CYP2B6 in the liver. Moreover, ATF5 and its target gene CYP2B6 are induced under different stress conditions, suggesting a new potential mechanism to adapt hepatic cytochrome P450 expression to diverse endobiotic/xenobiotic harmful stress.