Determining the molecular basis for the observed species differences in the xenobiotic-mediated induction of cytochrome 3A (CYP3A) gene expression has become one of the biggest dilemmas of the modern era in toxicology. Recently, a novel orphan nuclear receptor, termed pregnane X receptor (PXR), has been implicated to play a key role in the regulation of CYP3A genes by xenobiotics. PXR is capable of binding to and activating transcription from specific response elements found in the CYP3A gene promoter from multiple species. Notably, compounds that are known to induce CYP3A selectively in human, mouse, rat, or rabbit also activate the corresponding PXR. Pregnenolone 16alpha-carbonitrile, a known CYP3A inducer in rodents, is a very efficacious activator of mouse and rat PXR, whereas rifampicin, a known inducer of CYP3A in humans and rabbits, is a very efficacious activator of human and rabbit PXR. Likewise, selective activators of PXR also induce CYP3A gene expression in the corresponding species. Orthologous receptors from human, mouse, rat, and rabbit have been cloned and characterized and share approx. 95% identity in their DNA binding domains. By contrast, they share only 75-80% identity in their amino acid sequences in the ligand-binding domain. Together, these data suggest that PXR is a critical regulator of CYP3A gene expression and activation of PXR is predictive of CYP3A induction. Furthermore, sequence differences in the ligand-binding domain, and not the DNA binding domain, appear to serve as the molecular basis for the species differences in CYP3A induction observed in vivo.