Cibacron Blue, a widely used ligand for affinity chromatography, is a potent inhibitor of NAD(P)H:(quinone-acceptor) oxidoreductase (EC 126.96.36.199) (quinone reductase). This property has been exploited to purify quinone reductase, to identify its nucleotide-binding site, and to obtain diffraction-grade crystals of this enzyme [Prochaska, H. J. (1988) Arch. Biochem. Biophys. 267, 529-538; Ysern, X., & Prochaska, H. J. (1989) J. Biol. Chem. 264, 7765-7767]. To define the structural region(s) of the dye responsible for its inhibitory potency, Cibacron Blue was synthesized and the dye, its synthetic intermediates, and some analogues of these intermediates were crystallized as novel trialkylamine or choline salts. These compounds were characterized by proton NMR and mass spectrometry, and their inhibitory potencies were measured. Only two of the four ring systems of the Cibacron Blue molecule are required for potent inhibition. Acid Blue 25 [1-amino-4-(phenylamino)anthraquinone-2-sulfonic acid] is an inhibitor (Ki = 22 nM) almost as potent as Cibacron Blue (Ki = 6.2 nM). However, removal of any of the three substituents on the anthraquinone ring of Acid Blue 25 markedly reduced inhibitory potency. These results are consistent with the proposal that Cibacron Blue is primarily a mimic for the ADP fragment of mono- and dinucleotides. The difference absorption spectrum of the Acid Blue 25-quinone reductase complex was very different from that of the complex with Cibacron Blue. In contrast to other compounds tested, Procion Blue M-3GS, the electrophilic dichlorotriazine precursor of Cibacron Blue, was an irreversible inhibitor of quinone reductase (KD = 16 nM, k3 = 0.03 min-1), and the inactivation was blocked by Cibacron Blue, a monochlorotriazine.