The molecular identity of the thyroid microsomal antigen was investigated by Western blot and immunoprecipitation using sera from eight patients with autoimmune thyroid disease. All sera had high microsomal antibody titers by hemagglutination test and ELISA; only one had thyroglobulin (Tg) antibody. In an immunoprecipitation study using Triton X-100-solubilized 125I-labeled microsomes, all of the sera precipitated a 107K protein. However, this 107K protein was visualized by only three patients' sera by Western blot analysis under reducing conditions. In Western blots run under nonreducing conditions, four sera, including the three sera mentioned above, recognized poorly defined large mol wt proteins. One serum which also had Tg antibody recognized additional bands by both immunoprecipitation and Western blot. Preincubation of serum with 100 micrograms/ml Tg or 100 mU/ml bovine TSH did not alter the binding of antibody to 107K protein in Western blot analysis. The 107K protein was not visualized in Western blots of liver and kidney microsomes using patients' sera. Experiments were performed to characterize the difference between one serum (no. 1) which visualized the 107K band in Western blots and a serum (no. 4) which did not. Reactivity at different dilutions was compared by ELISA and Western blot studies. Serum 1 recognized the 107K protein even at a 1:3200 dilution, which gave a lower optical density value than a 1:200 dilution of serum 4 in ELISA, while serum 4 failed to recognize the 107K band at any dilution. An affinity gel prepared from serum 4 immunoglobulin G linked to agarose [Reacti-Gel (6 X)] removed from Triton X-100-solubilized microsomes the 107K protein which patient 1 serum recognized by Western blot analysis under reducing conditions. The data indicate that serum 4 can recognize the 107K protein under nondenaturing conditions. These data indicate that the 107K protein described here is the microsomal antigen, and that may associate with another protein or maintain a unique conformation due to disulfide bonds present in the native state. In addition, the 107K protein includes at least two antigen epitopes and different patients have different antibodies or different populations of antibodies against these epitopes.