Human prostate cancer is characterized by an early and near-universal loss of expression of the phase 2 enzyme glutathione S-transferase-pi (GSTP1). We hypothesize that a mechanism-based prostate cancer preventive strategy could involve induction of phase 2 enzymes within the prostate to compensate for the loss of GSTP1 expression. NAD[P]H:(quinone-acceptor) oxidoreductase (quinone reductase or QR) enzymatic activity, a surrogate of phase 2 enzyme response, was measured after treating the human prostate cancer cell line LNCaP with known phase 2 enzyme-inducing agents from 10 distinct chemical classes. QR enzymatic activity was assayed in microtiter plates using the menadione-coupled reduction of tetrazolium dye. Degree of induction was expressed as fold-increase over control and corrected for toxicity. Compounds were also tested in LNCaP-5-aza-C, an LNCaP subline selected in 5-aza-cytidine that expresses GSTP1, and in the human liver cell line HepG2. LNCaP showed robust induction of QR enzymatic activity after treatment with a subset of the phase 2 enzyme-inducing agents. All Michael acceptors were effective at inducing QR activity in LNCaP. Some phenolic antioxidants, heavy metal salts, and quinones also significantly increased QR activity, although inducer potency varied widely within these classes of compounds. Some of the isothiocyanates, mercaptans, bifunctional inducers, and trivalent arsenicals also produced modest QR induction, but peroxides and dithiolethiones were inactive. LNCaP-5-aza-C and LNCaP responded similarly to all compounds, but the pattern of response for HepG2 differed significantly. The differences in QR responsiveness between the prostate cell lines and HepG2 suggest that prostate tissues may have a unique pattern of response to phase 2-inducing agents distinct from other tissue types. Our data suggest that measurement of QR induction in prostate cancer cell lines may help identify potential cancer chemopreventive agents effective in the prostate.