To better understand the role of transcription factor NF-E2-related factor (NRF) 2 in the human and its contribution to cancer chemoprevention, we have knocked down its negative regulators, Kelch-like ECH-associated protein 1 (KEAP1) and broad-complex, tramtrack and bric à brac and cap'n'collar homology 1 (BACH1), in HaCaT keratinocytes. Whole-genome microarray revealed that knockdown of KEAP1 resulted in 23 messenger RNAs (mRNAs) being up-regulated > or = 2.0-fold. mRNA for aldo-keto reductase (AKR) 1B10, AKR1C1, AKR1C2 and AKR1C3 were induced to the greatest extent, showing increases of between 12- and 16-fold, whereas mRNA for glutamate-cysteine ligase catalytic and modifier subunits, NAD(P)H:quinone oxidoreductase-1 and haem oxygenase-1 (HMOX1) were induced between 2.0- and 4.8-fold. Knockdown of BACH1 increased HMOX1 135-fold but induced the other genes examined to a maximum of only 2.7-fold. Activation of NRF2, by KEAP1 knockdown, caused a 75% increase in the amount of glutathione in HaCaT cells and a 1.4- to 1.6-fold increase in their resistance to the electrophiles acrolein, chlorambucil and cumene hydroperoxide (CuOOH), as well as the redox-cycling agent menadione. Inhibition of glutathione synthesis during KEAP1 knockdown, by treatment with buthionine sulfoximine, abrogated resistance to acrolein, chlorambucil and CuOOH, but not to menadione. In contrast, knockdown of BACH1 did not increase glutathione levels or resistance to xenobiotics. Knockdown of NRF2 in HaCaT cells decreased glutathione to approximately 80% of normal homeostatic levels and similarly reduced their tolerance of electrophiles. Thus, the KEAP1-NRF2 pathway determines resistance to electrophiles and redox-cycling compounds in human keratinocytes through glutathione-dependent and glutathione-independent mechanisms. This study also shows that AKR1B10, AKR1C1 and AKR1C2 proteins have potential utility as biomarkers for NRF2 activation in the human.