Thyroid peroxidase (TPO) was purified from each thyroid of 10 patients (experiment 1) and 4 patients (experiment 2) with toxic diffuse goiter by a simplified method with monoclonal anti-TPO antibody-assisted immunoaffinity column chromatography. The final preparations were used to measure the heme concentration based on the cyanide difference spectrum, and to determine the Km and kcat values from double reciprocal plots in the assay employing guaiacol and iodide as the second substrates. The heme-based specific activities of TPO purified from thyroids in experiment 1 were higher than those in experiment 2, which were probably impaired by freezing-thawing, and those of porcine TPO previously reported. There were some differences in the kinetic properties between experiment 1 and experiment 2, but the individual differences within each group were relatively small, the values for CV (= SD/mean) being 0.16-0.48.