Rat liver NAD(P)H:quinone oxidoreductase cDNA was cloned and expressed in a eukaryotic cell expression plasmid containing a cytomegalovirus (CMV) promoter. Transient expression of enzyme activity and RNA transcription were measured in COS7 cells. The expressed quinone reductase has kinetic properties similar to the rat liver enzyme and is inhibited by dicourmarol, a known inhibitor of NAD(P)H:quinone oxidoreductase. Site-directed mutagenesis experiments carried out using this expression system revealed possible regions involved in NAD(P)H binding.