Derivatives of imidazole-2-thiones block reactions catalyzed by thyroid peroxidase (TPX) and the closely related lactoperoxidase (LPX), and this property is used therapeutically to treat hyperthyroidism. The reactions of a series of benzimidazoline-2-thiones with chemical and enzymatic oxidants were investigated to probe systematically the mechanism of inhibition. Oxidation of benzimidazoline-2-thione (I) and 1-methylbenzimidazoline-2-thione (II) with 3-chloroperbenzoic acid (PBA) yielded reaction products and stoichiometry consistent with benzimidazole-2-sulfenic acids as reactive intermediates. The N,N'-disubstituted nature of 1,3-dimethylbenzimidazoline-2-thione (III) precludes sulfenic acid formation by tautomerization, and the oxidation of III with PBA yielded products and stoichiometry that were consistent with a benzimidazole-2-sulfonyl ylide as the reactive intermediate. I and II are suicide inhibitors of LPX and TPX, but III was found to inhibit only peroxidase-catalyzed iodination reactions by an alternate substrate mechanism. These results provide support for the hypothesis that imidazole-2-sulfenic acids are important reactive intermediates in the suicide inactivation of TPX and LPX and relate the chemical reactivity of the inhibitor with both the potency and mechanism of inhibition. These results suggest that 1,3-disubstituted thiourea derivatives represent a new class of potential antihyperthyroid drugs that block TPX-catalyzed tyrosine iodination but do not cause irreversible enzyme inactivation.