Retinoid-related orphan nuclear receptors (RORs) α and γ (NR1F1, - 3) are highly expressed in liver, adipose tissue, thy-mus, and brain and are involved in many physiological processes, such as circadian rhythm and immune function. Enzymes in the cytochrome P450 2C subfamily metabolize many clinically important drugs and endogenous compounds, such as the anticancer drug paclitaxel and arachidonic acid, and are highly expressed in liver. Here, we present the first evidence that RORs regulate the transcription of human CYP2C8. Over-expression of RORa and RORy in HepG2 cells significantly enhanced the activity of the CYP2C8 promoter but not that of the CYP2C9 or CYP2C19 promoters. Computer analyses, promoter deletion studies, gel shift assays, and mutational analysis identified an essential ROR-responsive element at - 2045 base pairs in the CYP2C8 promoter that mediates ROR transactivation. Adenoviral overexpression of ROR α and γ significantly induced endogenous CYP2C8 transcripts in both HepG2 cells and human primary hepatocytes. Knockdown of endogenous RORα and - γ expression in HepG2 cells by RNA interference decreased the expression of endogenous CYP2C8 mRNA by ∼50%. These data indicate that RORs transcriptionally up-regulate CYP2C8 in human liver and, therefore, may be important modulators of the metabolism of drugs and physiologically active endogenous compounds by this enzyme in liver and possibly extrahepatic tissues where RORs are expressed. Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics.