The pH dependence of the redox behavior of anthranilate hydroxylase from Trichosporon cutaneum in its uncomplexed and anthranilate-complexed forms, as well as the effects on the reduction potential, at pH 7.4, of enzyme in complex with 3-methylanthranilate, salicylate, 3-acetylpyridine adenine dinucleotide phosphates, and azide plus anthranilate, is described. At pH 7.4 the midpoint potential of uncomplexed enzyme (EFlox/EFlredH-) is -0.229 V vs SHE, close to that of free flavin. The aromatic substrates and effector all shift the midpoint potential value in a positive direction by 0.068-0.100 V. This shift results in thermodynamically more favorable reduction of the substrate/effector-complexed enzyme by NADPH. Consistent with thermodynamic considerations, the aromatic substrates (or effector) are bound to the reduced enzyme 2-4 orders of magnitude more tightly than to the oxidized enzyme. The tighter binding of the substrate to the two-electron-reduced enzyme may be related to the double hydroxylation reaction performed by this enzyme, which is a more complex reaction than is carried out by typical flavoprotein hydroxylases. The acetylpyridine nucleotides appear to have no significant regulatory role.