The transcription factors that bind to EpRE's play a key role in the regulation of phase II genes. In this study, we examined whether c-Jun, a partner of Nrf2 in binding to EpRE's, requires phosphorylation by JNK for binding and transcriptional activation. We used chromatin immunoprecipitation assays to measure the recruitment of transcription factors to EpRE sequences in NQO2, GCLC, and GCLM; Western analysis for phosphorylation of JNK; and EpRE-driven reporters along with a JNK-specific inhibitor peptide to determine the potential importance of c-Jun phosphorylation. Human bronchial epithelial (HBE1) and human hepatoma (HepG2) cells were exposed to 4-hydroxy-2-nonenal (HNE), and differences in the regulation of the same EpRE sequences were examined. We found that binding of c-Jun to EpRE sequences increased subsequent to HNE exposure in HepG2 cells; however, in HNE-exposed HBE1 cells, the binding of only phosphorylated c-Jun to the three EpRE sequences increased. Despite the increase in binding of phosphorylated c-Jun, reporter assays for EpRE's showed that inhibition of c-Jun phosphorylation had variable effects on basal and HNE-induced transcription of GCLC and GCLM in HBE1 cells. Thus, in terms of its role in mediating HNE induction of EpRE-mediated transcription, c-Jun seems to be a partner of Nrf2 and, whereas its phosphorylated form may predominate in one cell type versus another, the effects of phosphorylation of c-Jun on transcription can vary with the gene. This contrasts markedly with the well-established requirement for phosphorylation of c-Jun in the activation of AP-1/TRE-mediated transcription.