Using a panel of 20 well-characterized mouse monoclonal antibodies (mAbs) to native human thyroglobulin (Tg), the fine specificities of autoantibody responses in human autoimmune thyroiditis patients were compared to naturally occurring autoantibodies from healthy individuals. The antigenic determinants recognized by the human autoantibodies were assessed using competitive binding assays in which murine mAbs were inhibited by human autoantibodies. All determinants of human Tg recognized by the mouse were also responded to by the human subjects. Autoantibodies from adult and juvenile patients with chronic lymphocytic thyroiditis recognized principally 10 of 19 determinants defined by the panel of murine mAbs. The mAbs which define these 10 determinants do not react with Tg from seven other mammalian species; therefore, autoantibody responses in individuals with thyroiditis are directed primarily to species specific determinants of human Tg. These same determinants on human Tg were the principal ones recognized by the mouse following immunization. Particular patterns of fine specificities were not inherited from parents by probands or their siblings, even if identical twins. Naturally occurring Tg autoantibodies from healthy subjects recognized mainly thyroxine-containing cross-reactive determinants.