Different antigenic determinants on thyroid microsomal antigen (MAg) could induce different antibodies, which, in turn, could have differing importance in the pathogenesis of autoimmune thyroid disease. In this study we demonstrated three types of microsomal antibodies (MAbs) present in serum of patients with autoimmune thyroid disease by Western blot analysis. These MAbs reacted with native, denatured, and denatured and reduced MAg. Methods for detecting the MAbs were developed, and the clinical significance of these Abs was analyzed in 197 patients with thyroid disorders. For measurement of Ab against native and denatured MAg, an enzyme-linked immunosorbent assay, in which wells were coated with untreated or sodium dodecyl sulfate-denatured MAg, was used. For measurement of Ab against denatured and reduced MAg, 6% SDS-polyacrylamide gel electrophoresis followed by Western blot analysis was used. Native MAb enzyme-linked immunosorbent assay values correlated highly with microsomal hemagglutination titers, but patients with high titers of Ab against native MAg did not necessarily have Ab against denatured MAg or reduced MAg. Abs against denatured MAg or denatured and reduced MAg were found in patients with Hashimoto's disease (28.8% and 13.6%) and Graves' disease (22.4% and 11.2%). The percentage of patients with positive denatured and reduced MAb was higher in Graves' disease patients who had a longer sick interval or who developed hypothyroidism after radioiodine treatment. This study provides the first clear demonstration that MAbs are heterogeneous. The data suggest that antibodies against denatured or denatured and reduced MAg may be related to destruction of the thyroid gland.