Reverse transcriptase-polymerase chain reaction was used to amplify a partial cDNA from rabbit lung mRNA that shared 77% protein sequence identity with the mouse pregnane X receptor (PXR). Rapid amplification of cDNA ends from a rabbit kidney lambdaZAP expression library resulted in the isolation of overlapping cDNAs spanning the complete coding sequence. The deduced amino acid sequence of 411 residues exhibited 79% overall amino acid identity with human PXR and 77% identity with mouse PXR. Based on this protein sequence relationship and a similar degree of conservation exhibited by the mouse and human PXR orthologs, the cDNA appears to encode the rabbit PXR ortholog. 5'-rapid amplification of cDNA ends performed on an adaptor-ligated cDNA library from rabbit liver revealed the presence of an alternate mRNA, which differed at the 5'-terminus. RNase protection assays indicated that the alternate mRNA was expressed at >50-fold lower levels in rabbit kidney and liver. Rifampicin treatment of CV-1 cells cotransfected with a rabbit PXR expression plasmid and a luciferase reporter construct containing two copies of the DR3 enhancer from CYP3A23 produced a 6-fold induction of luciferase activity. In contrast, rat PXR was not responsive to this antibiotic under the same conditions. Pregnenolone 16alpha-carbonitrile was an efficacious activator of rat PXR, but failed to significantly activate rabbit PXR at equivalent concentrations. These results indicate that the ligand activation profile of rabbit PXR is distinct from rat PXR and more closely resembles that of human PXR. The rabbit PXR activation profile is consistent with the cytochrome P450 (P450) 3A6 induction profile in rabbits.