Peroxisome proliferators activate nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha) and enhance the transcription of several genes in liver. We report here that synthetic PPARalpha ligands Wy-14,643, ciprofibrate, clofibrate, and others induce the nuclear translocation of constitutive androstane receptor (CAR) in mouse liver cells in vivo. Adenoviral-enhanced green fluorescent protein-CAR expression demonstrated that PPARalpha synthetic ligands drive CAR into the hepatocyte nucleus in a PPARalpha- and PPARbeta-independent manner. This translocation is dependent on the transcription coactivator PPAR-binding protein but independent of coactivators PRIP and SRC-1. PPARalpha ligand-induced nuclear translocation of CAR is not associated with induction of Cyp2b10 mRNA in mouse liver. PPARalpha ligands interfered with coactivator recruitment to the CAR ligand binding domain and reduced the constitutive transactivation of CAR. Both Wy-14,643 and ciprofibrate occupied the ligand binding pocket of CAR and adapted a binding mode similar to that of the CAR inverse agonist androstenol. These observations, therefore, provide information for the first time to indicate that PPARalpha ligands not only serve as PPARalpha agonists but possibly act as CAR antagonists.