Measurements of thyrotropin and of total and free thyroxine and triiodothyronine are widely used diagnostic methods for thyroid function evaluation. However, some serum samples will demonstrate a nonspecific binding with assay reagents that can interfere with the measurement of these hormones. Several recent case reports have described the presence of such interferences resulting in reported abnormal concentrations of thyroid hormones inconsistent with the patient's thyroid state. Circulating thyroid hormone autoantibodies, described in thyroid and nonthyroid disorders, are an important class of interference factor and can bind to hormone tracers used in various immunoassays. Two additional categories of interfering antibodies may particularly interfere within two-site immunoassays for thyrotropin. These include heterophile antibodies, especially human anti-mouse antibodies, and rheumatoid factors, which can cause interferences by immunoglobulin aggregation and (or) cross-linking of both capture and signal antibodies. Here we review the nature of these disturbances; their occurrence, prevalence, and detection; and the clinical consequences of the failure to recognize such interference.