BACKGROUND AND PURPOSE: Rilpivirine and etravirine are second-generation non-nucleoside reverse transcriptase inhibitors (NNRTIs) indicated for the treatment of HIV/AIDS. The constitutive androstane receptor (CAR) regulates the expression of genes involved in various biological processes, including the transport and biotransformation of drugs. We investigated the effect of rilpivirine and etravirine on the activity of the wild-type human CAR (hCAR-WT) and its hCAR-SV23 and hCAR-SV24 splice variants, and compared it with first-generation NNRTIs (efavirenz, nevirapine, and delavirdine). EXPERIMENTAL APPROACH: Receptor activation, ligand-binding domain (LBD) transactivation, and co-activator recruitment were investigated in transiently transfected, NNRTI-treated HepG2 cells. Nuclear translocation of green fluorescent protein-tagged hCAR-WT and CYP2B6 gene expression were assessed in NNRTI-treated human hepatocytes. KEY RESULTS: Rilpivirine and etravirine activated hCAR-WT, but not hCAR-SV23 or hCAR-SV24, and without transactivating the LBD or recruiting steroid receptor coactivators SRC-1, SRC-2, or SRC-3. Among the first-generation NNRTIs investigated, only efavirenz activated hCAR-WT, hCAR-SV23, and hCAR-SV24, but none of them transactivated the LBD of these receptors or substantively recruited SRC-1, SRC-2, or SRC-3. Rilpivirine, etravirine, and efavirenz triggered nuclear translocation of hCAR-WT and increased hCAR target gene (CYP2B6) expression. CONCLUSION AND IMPLICATIONS: NNRTIs activate hCAR-WT, hCAR-SV23, and hCAR-SV24 in a drug-specific and isoform-selective manner. The activation occurs by a mechanism that does not appear to involve binding to the LBD or recruitment of SRC-1, SRC-2, or SRC-3