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Human constitutive androstane (or active) receptor (hCAR), a member of the nuclear receptor superfamily NR1I3, regulates the expression of several genes that are mainly involved in the metabolism of endogenous and xenobiotic compounds (e.g., CYP2B6, CYP3A4, and UGT1A1). We found four novel splice variants in the ligand-binding domain (LBD) of hCAR (NCBI reference sequence, NM_005122; designated SV0 herein). The variants designated SV1 and SV2 contained in-frame 12- and 15-base pair (bp) insertions, respectively. SV3 carried both of the insertions, and SV4 contained an in-frame 117-bp deletion. The insertion site of SV1 is located in the alpha6 helix of hCAR LBD, which makes up the ligand-binding cavity, and that of SV2 is located in the highly conserved loop between helices alpha8 and alpha9. SYBR Green real-time reverse transcription-polymerase chain reaction analysis of each splice variant revealed that the hepatic expression of SV2 was almost comparable with that of SV0 (approximately 40%), whereas other variants accounted for 6 to 10% of the total hCAR transcripts. In the reporter gene assays employing the phenobarbital-responsible enhancer module (PBREM) from CYP2B6 and UGT1A1 genes, the splice variants, except for SV1, were inactive, whereas SV1 transactivated the CYP2B6 PBREM but not the UGT1A1 PBREM reporter. A nuclear translocation assay in rat hepatocytes revealed that all the splice variants lack the responsiveness toward phenobarbital and 6-(4-chloropheny-l)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO) in terms of the ligand-dependent nuclear translocation. Further characterization, such as the identification of specific ligands, will help elucidate physiological implication of these hCAR splice variants.