Ischemia/reperfusion injury increases the expression of bioactive heparin-binding epidermal growth factor-like growth factor (HB-EGF) in the rat kidney, suggesting that oxidant stress or cell injury related to oxidant stress might affect HB-EGF expression in the injured renal parenchyma. We utilized a nontransformed rat renal epithelial cell line (NRK-52E cells) to investigate whether reactive oxygen species induced transcriptional activation of HB-EGF mRNA. Hypoxia/reoxygenation increased HB-EGF expression in NRK-52E cells, and at concentrations that induced sublethal cell injury, hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) increased HB-EGF mRNA expression 4.7-fold. The free radical scavengers, dimethylthiourea and N-acetylcysteine inhibited HB-EGF mRNA induction. In contrast, another free radical scavenger, pyrrolidine thiocarbamate (PDTC), augmented H<sub>2</sub>O<sub>2</sub>-mediated HB-EGF expression. Since PDTC has been reported to augment AP-1-mediated transcriptional activation, we utilized an electrophoretic mobility shift assay to confirm that H<sub>2</sub>O<sub>2</sub> administration to NRK-52E cells did increase nuclear extract DNA-binding activity to a consensus AP-1 sequence. Using a CAT reporter assay coupled to the proximal 2,000 bp of the human HB-EGF 5′-untranslated region, we determined that H<sub>2</sub>O<sub>2</sub> administration increased CAT activity 5.5-fold. Truncation or deletion mutations of a putative AP-1-binding site reduced the H<sub>2</sub>O<sub>2</sub>-stimulated activity by >60%, and there was increased DNA binding of nuclear extracts from H<sub>2</sub>O<sub>2</sub>-treated cells to a 24-bp oligonucleotide containing this putative AP-1 site. Anti- fos and jun antibodies inhibited this binding, and there was no binding to an oligonucleotide in which the putative AP-1 site was mutated.The site of the residual activation was found to exist in the most proximal 5′-untranslated region (–121 to +60), which contains two putative SP1 sites. Timing and localization of AP-1-binding activity from nuclear extracts from the post-ischemic tissue correlated with HB-EGF mRNA expression. Therefore, in renal epithelial cells, oxidant stress increases HB-EGF expression, which appears to be mediated in part by an increase in AP-1 binding. This activation may play an important role in the induction of HB-EGF mRNA in response to tissue injury and may regulate early stages of recovery following ischemic damage.