The pregnane X receptor (PXR) is a nuclear receptor significantly involved in the transcriptional regulation of drug-metabolizing enzymes and transporters. Interestingly, certain PXR ligands such as rifampin have been shown to readily induce human and rabbit but not rodent members of the cytochrome P450 3A. Because drugs of divergent chemical structures seem to be similarly affected, we hypothesized that specific amino acid residue(s) or domains in rat PXR affect receptor activation by certain human PXR ligands. To identify such a domain(s), an array of human-rat and rat-human chimeric PXR cDNAs in a tandem head-to-tail configuration were created using a random chimeragenesis method. Pharmacological characterization of these chimeras revealed a discreet segment within the ligand-binding domain of rat and human PXR to be essential for the rifampin effect. Within this region, the corresponding residues Leu308 and Phe305 of human and rat PXR, respectively, were found to be important for rifampin activation. Homology modeling derived from the recently determined crystal structure of human PXR indicates that these amino acids are located within or neighboring the flexible loop that forms part of the pore to the ligand-binding cavity. Rifampin, paclitaxel, and hyperforin sensitivity was conferred to rat PXR when Phe305 was converted to leucine, whereas attenuation of sensitivity was observed when Leu308 of human PXR was replaced with phenylalanine. Accordingly, our data provide compelling new insight into the importance of the amino acids comprising the pore to the ligand-binding cavity as a critical modulator of PXR response.