The constitutive androstane receptor (CAR) regulates mouse and human CYP2B genes through binding to the direct repeat-4 (DR4) motifs present in the phenobarbital-responsive enhancer module (PBREM). The preference of PBREM elements for nuclear receptors and the extent of cross-talk between CAR and other nuclear receptors are currently unknown. Our transient transfection and DNA binding experiments indicate that binding to DR4 motifs does not correlate with the activation response and that mouse and human PBREM are efficiently 'insulated' from the effects of other nuclear receptors despite their substantial affinity for DR4 motifs. Certain nuclear receptors that do not bind to DR4 motifs, such as peroxisome proliferator-activated receptor-alpha and farnesoid X receptor, can suppress PBREM function via a coactivator-dependent process that may have relevance in vivo. In competition experiments, mouse PBREM is clearly more selective for CAR than human PBREM. Pregnane X, vitamin D, and thyroid hormone receptors can potentially compete with human CAR on human PBREM. In contrast to the selective nature of PBREM, CYP3A enhancers are highly and comparably responsive to CAR, pregnane X receptor, and vitamin D receptor. In addition, the ligand specificities of human and mouse CAR were defined by mammalian cotransfection and yeast two-hybrid techniques. Our results provide new mechanistic explanations to several previously unresolved aspects of CYP2B and CYP3A gene regulation.