Inorganic arsenic is an environmental contaminant and a known human carcinogen. Arsenite (trivalent) is thiol-reactive and more toxic than arsenate (pentavalent). Low levels of arsenite cause oxidative stress in cells and evoke cellular defense responses that lead to the up-regulation of stress-response proteins such as heme oxygenase-1 (HO-1) and the molecular chaperone Hsp70. HO-1 expression is strongly induced in response to a variety of agents in addition to arsenic. Previous studies have implicated the transcriptional enhancers Nrf2 (1), AP-1, and HSF1 in the arsenite-induced murine ho-1 gene activation, although their signaling pathways and relative contributions have not been fully elucidated. We used wild-type and Nrf2-deficient murine embryonic fibroblasts to investigate the induction of HO-1 in response to low levels of arsenite. We found that Nrf2-/- cells are more sensitive than Nrf2+/+ cells to arsenite toxicity, and that Nrf2 plays a crucial role in the early activation of ho-1 transcription. In Nrf2-/- cells, ho-1 gene activation, mainly mediated via AP-1 and HSF1, increased slowly, and during hours 4-12, the HO-1 protein levels were only half of those attained in the Nrf2+/+ cells. Our results suggest that genistein-sensitive tyrosine kinase and JNK activities play significant roles in the AP-1 signaling pathway, but not in the Nrf2 or HSF1 activation pathways. Inhibitors of ERK1/2, p38MAPK, and PKC had negligible effects on the arsenite-induced HO-1 expression levels. Our findings provide the first evidence that Nrf2 plays an important role in early HO-1 induction and in protecting the cells from arsenite toxicity, and that the Nrf2-independent pathway contributes to long-lasting activation of the ho-1 gene.