The constitutive androstane receptor (CAR, NR1I3) regulates the expression of genes involved in all phases of xenobiotic metabolism. Alternative splicing of the human CAR transcript yields an array of unique mRNAs. One form, termed CAR2, contains an additional 4 amino acids (SPTV) that are predicted to reshape the ligand-binding pocket. The current studies demonstrate a marked, ligand-independent, CAR2-mediated transactivation of reporters containing optimal DR-3, DR-4 and DR-5 nuclear receptor response elements, and reporters derived from the natural CYP2B6 and CYP3A4 gene promoters. Over expression of RXRα was critical for achieving these effects. CAR2 interaction with SRC-1, assessed by mammalian 2-hybrid experiments, was similarly dependent on RXRα. Mutagenesis of S233 (SPTV) to an alanine residue yielded a receptor possessing higher constitutive activity. Alternatively, mutating S233 to an aspartate residue drastically reduced CAR2’s transactivation capacity, a result that correlates to their ability to interact with RXRα, and to recruit SRC-1 in an inverse-agonist regulated manner. Together, these results demonstrate a robust RXRα-dependent recruitment of coactivators and transactivation by CAR2. In addition, CAR2’s novel dose response to clotrimazole and androstanol when compared to the reference form of the receptor suggests that CAR2 may be regulated by a unique set of ligands.