Phenobarbital is a lipophilic molecule used as a sedative and antiepileptic drug that elicits a multitude of effects in the liver, including gross liver enlargement, hepatocyte hypertrophy, and induced expression of drug-metabolizing enzymes and other liver-specific genes. The constitutive androstane receptor (CAR; NR1I3) and to a lesser extent the pregnane X receptor (PXR; NR1I2) are responsible for mediating induction of many phenobarbital-responsive genes. However, CAR-mediated transcriptional control of some genes is critically dependent on hepatocyte nuclear factor 4 alpha (HNF-4alpha; NR2A1), which itself regulates multiple liver-specific genes involved in hepatic growth, metabolism, and differentiation. We studied the effects of phenobarbital on HNF-4alpha expression in hepatocytes and provide evidence that HNF-4alpha nuclear expression is regulated in response to phenobarbital. Real-time polymerase chain reaction analyses revealed that HNF-4alpha mRNA is modestly up-regulated by phenobarbital. In addition, nuclear expression of HNF-4alpha protein is significantly elevated 3 hours after the administration of phenobarbital in wild-type, CAR-/-, and CAR-/-/PXR-/- mice. In vitro analysis revealed that phenobarbital-induced HNF-4alpha expression is both time- and dose dependent. In addition, the phosphatase inhibitor okadaic acid and the Ca2+/calmodulin-dependent protein kinase II inhibitor KN62 block nuclear induction of HNF-4alpha by phenobarbital. Furthermore, HNF-4alpha nuclear expression is enhanced by inhibition of cyclic AMP-dependent protein kinase A. In conclusion, induced nuclear expression of HNF-4alpha and CAR is an integral part of the phenobarbital response, aimed at coordinated regulation of genes involved in drug metabolism and detoxification as well as maintenance of liver function.