The reaction between o-, m-, and p-methoxyphenols and 3-methyl-2-benzothiazolinone hydrazone (MBTH) is studied in the presence of horseradish peroxidase (HRP) and H(2)O(2 )as oxidative agent. The findings indicate that enzyme (H2O2 oxidoreductase; EC 1.11.1.7) catalyzes an oxidative coupling reaction between MBTH and phenols which produces azo dye compounds.On the basis of kinetic parameters and optimum pH values, a mechanism in which both MBTH and phenols seem to be activated by the HRP for achieving the oxidative coupling is proposed. Furthermore, in the current study, we have evaluated the possibility that these azo dyes may be useful in the measurement of peroxidase activity. The method is based on the observed increase in the absorbance at 502 nm (8,355 cm(-1) M-1 of extinction molar coefficient) due to the formation of a red azo dye compound resulting from the peroxidase-catalyzed oxidative coupling of MBTH and o-methoxyphenol (guaiacol). Using this assay system HRP can be determined in picomolar levels by a fixed time method.