In the liver, sterol 27-hydroxylase (CYP27) participates in the classic and alternative pathways of bile acid biosynthesis from cholesterol (Chol). In extrahepatic tissues, CYP27 converts intracellular Chol to 27-hydroxycholesterol (27OH-Chol), which may regulate the activity of 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA-R). This study attempts to better define the role of CYP27 in the maintenance of Chol homeostasis in hepatic and extrahepatic cells by overexpressing CYP27 in Hep G2 cells and Chinese hamster ovary (CHO) cells through infection with a replication-defective recombinant adenovirus encoding for CMV-CYP27. After infection, CYP27 mRNA and protein levels increased dramatically. CYP27 specific activity also increased two- to fourfold in infected cells (P < or = 0.02), with a marked increase in conversion of [(14)C]Chol to [(14)C]27OH-Chol (approximately 150%; P < or = 0.01). Accumulation of 27OH-Chol in CHO cells was associated with a 50% decrease in HMG-CoA-R specific activity (P < or = 0.02). In infected Hep G2 cells, the significant increase in bile acid synthesis (46%; P < or = 0.006), which prevented the accumulation of intracellular 27OH-Chol, resulted in increased HMG-CoA-R activity (183%; P < or = 0.02). Overexpression of CYP27 in Hep G2 cells also increased acyl CoA-cholesterol acyltransferase (71%, P < or = 0.02) and decreased cholesteryl ester hydrolase (55%, P < or = 0.02). In conclusion, CYP27 generates different physiological responses depending on cell type and presence or absence of bile acid biosynthetic pathways.