To study the role of DT-diaphorase in menadione-mediated cytotoxicity, menadione-resistant cells were selected from P19 cells by stepwise increasing concentrations of menadione from 10 to 60, 120 or 300 microM without mutagenic pretreatment. Three isolated clones, K60, K120 and K300, were maintained in media containing 60, 120 or 300 microM menadione, respectively. The resistance of these cells to menadione, in order, was: K300 > K120 > K60 > P19 cells. K300 cells were the most resistant. Acquisition of resistance was associated with elevation in DT-diaphorase activity. Pretreatment of the resistant cells with 30 microM dicumarol at 37 degrees C for 30 min sensitized the resistant cells to menadione. When the resistant cells were maintained in the absence of menadione for 28 days, the resistance of K60 and K120 cells was lost. The lower degree of resistance was accompanied by a decrease in DT-diaphorase activity in the revertant cells. However, the resistance and the activity of DT-diaphorase in K300 cells were quite stable in the same period. These results support strongly that DT-diaphorase protects against menadione-induced oxidative stress.