Abstract The rate of reduction of 2,6-dichlorobenzenoneindophenol by xanthine oxidase plus its aldehydic or purine substrates is strikingly stimulated by the admixture of water-miscible organic solvents. In addition to acting as the electron acceptor in these assays, 2,6-dichlorobenzenoneindophenol is found to be a potent competitive inhibitor of xanthine oxidase with a KI of 1 x 10-6 M. The leuco form of this dye is a much less effective inhibitor, with a KI of 1.5 x 10-5 M. Dimethoxyethane and other water-miscible organic solvents reduce the inhibitory potency of 2,6-dichlorobenzenoneindophenol and thus give the appearance of activating the enzyme in this dye reduction assay. The inhibition of the enzyme by several other competitive inhibitors was not affected by dimethoxyethane. Dimethoxyethane, per se, is an inhibitor of xanthine oxidase. Its effects on the kinetics of this enzyme are notably complex. The reversal of the 2,6-dichlorobenzenoneindophenol inhibition by organic solvents appears best explained in terms of their effects on the solvent properties of the medium.