Nuclear translocation of constitutive androstane receptor (CAR) is a primary mechanism for the induction of cytochrome P450 genes by phenobarbital (PB). We have shown that exogenous expression of the p160 coactivator glucocorticoid receptor interacting protein-1 (GRIP1) in hepatocytes in vivo can mediate PB-independent nuclear accumulation of murine CAR (mCAR). To understand the mechanism of this PB-independent nuclear accumulation, we have examined the mCAR structural determinants of its GRIP1-mediated nuclear localization. Mutations of the xenobiotic response sequence (XRS), which had been shown to block PB-dependent nuclear translocation of human CAR in mouse hepatocytes in vivo, also blocked GRIP1-mediated nuclear accumulation of mCAR in mouse hepatocytes in vivo and further blocked nuclear localization in cultured HepG2 cells. A leucine 326 XRS mutant retained partial transcriptional activity, but mutations of three leucines in the XRS eliminated transcriptional activity in HepG2 cells, suggesting that the translocation function of the XRS overlaps with transcriptional functions. Mutation of the activation function 2 motif, by deletion of the C-terminal 8 amino acids, also reduced nuclear localization by both PB treatment and GRIP1 expression in hepatocytes in vivo, suggesting that either interaction with GRIP1 through this motif or active CAR was required for the nuclear localization. The localization of a DNA-binding domain mutant was essentially unchanged by coexpression of GRIP1, although without GRIP1 coexpression, this mutant expressed exhibited a more nuclear localization compared with wild type. The results are most consistent with a model in which GRIP1 interaction and activation of mCAR in the nucleus result in retention and accumulation of mCAR in the nucleus in untreated animals. The model requires that mCAR is constantly shuttling between the nucleus and cytoplasm even in untreated animals in which mCAR is predominantly cytoplasmic.