Purified DT-diaphorase can be assayed as either dicumarol-inhibitable NAD(P)H:menadione oxidoreductase or dicumarol-inhibitable NAD(P)H:dichlorophenolindophenol reductase. Both of these methods have been utilized to assay DT-diaphorase activity in tissue and cell homogenates. When DT-diaphorase activity was measured as dicumarol-inhibitable NADPH:dichlorophenolindophenol reductase in sonicates of two cell lines previously shown to not have any measurable activity of this enzyme, no enzymatic activity was detected. However, when the water-soluble bisulfite addition product of menadione was used as the electron acceptor, an artifactual activity for DT-diaphorase was detected in these cell lines. When another cell line was assayed utilizing menadione bisulfite, an apparent activity of about three times that found with dichlorophenolindophenol was measured, and thus, may overestimate DT-diaphorase activity in cells having activity. When menadione was used in place of menadione bisulfite, an artifactual DT-diaphorase activity was also detected, but was about one-half that obtained with menadione bisulfite. Polarographic determinations of the midpoint potentials for menadione and menadione bisulfite indicated that the latter compound was easier to reduce and may account for the greater apparent DT-diaphorase activity measured with this compound.