Recent reports have suggested that thyroid peroxidase (TPO) can be detected in the circulation of normal subjects and of patients with Graves' disease and we have attempted to confirm and extend these observations. A TPO radioimmunoassay with a sensitivity of 1 ng/mL was used to measure TPO in the sera from 20 normal subjects and 21 patients with Graves' disease. In addition, TPO was measured in serum samples from six normal subjects before and after oral TRH. We were unable to detect TPO in 46 out of the 47 sera studied (normals and autoimmune thyroid disease). In the one remaining serum (from a normal subject), low levels of TPO were apparently detected, but we demonstrated that this result was due to assay interference from TPO autoantibodies. Overall our studies suggest that (1) thyroid peroxidase is not detectable in normal subjects nor in TPO autoantibody negative patients with Graves' disease; (2) endogenous TPO autoantibodies can interfere in the TPO radioimmunoassay leading to false positive results; and (3) an acute increase of TSH in normal subjects does not result in TPO release into the circulation.