The activity of DT-diaphorase [NAD(P)H:(quinone-acceptor)oxidoreductase] is increased 7-fold in wild-type BALB/c 3T3T cells as they reach confluence and become density growth arrested. Harvesting and replating the cells at low density resulted in a loss of DT-diaphorase with a half time of 7 h, and removal of serum from high-density growth-arrested cells resulted in a decrease in DT-diaphorase with a half time of 3 days. Platelet-derived growth factor and insulin together, but not singly, maintain elevated DT-diaphorase levels in high-density growth-arrested BALB/c 3T3T cells. The increase in DT-diaphorase at high density diminished proportionately to the extent of transformation in four cell lines, 4NQO-3T3T, UV-3T3T, EJras-3T3T. and CSV3-1-3T3T. The most transformed cell line, CSV3-1-3T3T, showed no increase in DT-diaphorase at high density. Since there was no increase in DT-diaphorase mRNA in high-density growth-arrested wild-type BALB/c 3T3T cells compared to rapidly growing cells, the increase in DT-diaphorase activity at high density is most likely due to posttranslational events. High-density growth-arrested wild-type BALB/c 3T3 cells exhibited a greater sensitivity to growth inhibition by the antitumor quinone diaziquone [1,4-cyclohexadiene-1,4- dicarbamic acid, 2,5-bis(1-aziridinyl)-3,6-dioxo-, diethyl ether], which is metabolically activated by DT-diaphorase, than do low-cell-density, growth-arrested cells. The significance of the increase in DT-diaphorase at high cell density in normal cells and its loss in transformed cells may be related to the phenomenon of density-dependent growth inhibition in nontransformed but not in transformed cells.