The activity of the two-electron bioreductive enzyme DT-diaphorase (DTD) is induced by heat shock, hypoxic stress, oltipraz, and mitomycin C (MMC). Transcriptional induction is associated with nuclear factor binding to elements mediating immediate early response including AP-1, though the DTD mRNA peaks at 24 hr. Electrophoretic mobility shift assays revealed that nuclear protein extracts from hypoxia-, oltipraz-, and MMC-treated cells bound a specific oligonucleotide probe corresponding to the NF-kappa B transcriptional binding site in two human cancer cell lines, HT29 and HepG2. The binding activity for the NF-kappa B site was induced with a time-course similar to that of the induction of DTD, and was delayed in comparison to the induction of AP-1 binding proteins. The time-courses of the NF-kappa B binding response to MMC, oltipraz and hypoxic treatment were similar, and binding was most pronounced at 24 hr. All three stimuli were associated with the late appearance of a higher molecular weight complex in HT29 but not in HepG2 cells, suggestive of the participation of additional rel family proteins in DNA binding in this cell line. Competition experiments indicated that the bound protein complex was specific for the NF-kappa B binding site. An immunodepletion assay showed that in each case the bound complex consisted of a heterodimer of the NF-kappa B proteins p50 and p65. These data suggest that hypoxia, oltipraz and MMC may each induce the overexpression of DTD through a mechanism involving the NF-kappa B response element in the DTD 5'-flanking region, and support a role for this element in the control of detoxication responses to environmental changes.