Thyroid microsomal antigen and peroxidase (TPO) have a close intracellular anatomical relationship, especially in exocytotic vesicles. We considered that antibodies to microsomal antigen might react with TPO and therefore looked for the presence of antibodies against TPO in the serum of patients with autoimmune thyroid disease (AITD). TPO was prepared from Graves' thyroid glands, solubilized by n-octyl glucoside, and its activity was assayed by the guaiacol method. Control sera and sera with a positive microsomal hemagglutination test (MCHA(+) ) were assayed for their ability to precipitate TPO activity by incubation of sera with TPO and protein A. We identified MCHA(+) sera which caused precipitation of TPO activity, and the extent of precipitation was related to the amount of serum added. A significant correlation was present between this anti-peroxidase activity and microsomal antibodies titers, measured by a micro-ELISA method. Affinity columns prepared from immunoglobulins of MCHA(+) sera, coupled to Reacti-Gel (6X), bound TPO activity, whereas using control IgG the recovery in the unbound fraction was high. These data provide evidence of antibodies against thyroid peroxidase in the serum of patients with AITD and suggest a close link between microsomal antigen and thyroid peroxidase.