The nuclear constitutive active receptor (CAR) is a key transcription factor regulating phenobarbital (PB)-inducible transcription of various hepatic genes that encode xenobiotic/steroid-metabolizing enzymes. CAR is retained in the cytoplasm of noninduced livers and translocates into the nucleus after PB induction. HepG2 cells lack the capability of retaining CAR in the cytoplasm; thus, the receptor spontaneously accumulates in the nucleus. We have now cloned and characterized a tetratricopeptide repeat (TPR) protein, designated cytoplasmic CAR retention protein (CCRP), for its ability to accumulate the receptor in the cytoplasm of cotransfected HepG2 cells. CCRP directly interacts with the ligand-binding domain of CAR and mediates the formation of a cytoplasmic CAR-CCRP-90-kDa heat shock protein (hsp90) ternary complex. Simultaneous expression of fluorescent protein-tagged CAR and CCRP reveals their colocalization with tubulin in mouse liver in vivo. Thus, these results indicate that CCRP may be a component of the CAR-hsp90 complex and involved in retaining the receptor in the cytoplasm of both HepG2 cells and probably in vivo liver cells.