Assay of the anti-thyroperoxidase (TPO), anti-thyroglobulin (Tg), and anti-TSH receptor antibodies constitutes the basis of the laboratory diagnostic work-up for autoimmune thyroid diseases. However, although these antibodies have been routinely assayed in serum for more than 40 years with increasingly reliable methods, it still exists a wide analytical variability that influences their correct diagnostic use. We analysed the biochemical, physiopathological and clinical aspects of the thyroid antigen-anti body systems, and propose the following guidelines for using autoantibody tests and analytical assay methods: a) assay of anti-TPO with third generation ultrasensitive method should constitute the main test for the diagnosis of autoimmune thyroid diseases. Assay of anti-M antibodies should be considered obsolete because even purified microsome preparations contain traces of contaminants (Tg and other antigens) that make the test aspecific; b) assay of anti-Tg antibodies may be limited to patients with suspect AITD and negative for anti-TPO antibodies, and patients undergoing thyroglobulin assay, because anti-Tg autoantibodies may interfere in the assay of the molecule in the immunometric test; c) assay of the anti-TSH receptor antibodies is used to diagnose Basedow's disease, and atrophic chronic thyroiditis; d) it is appropriate to use normal reference values adjusted for age and gender.