The difference between stimulative and destructive thyrotoxicosis is usually identified by thyroidal uptake of radioactive iodine, but uptake is not always available. At present, there is no reliable in vitro method of differential diagnosis of thyrotoxicosis. We conducted an investigation of an in vitro method of differential diagnosis of thyrotoxicosis by inhibition assay using monoclonal antibodies that recognized an antigen (14kD) differing from thyroglobulin and thyroid peroxidase. The antigen is the soluble material present in the thyroid follicular space, and is detected by immunofluorescent study. We measured the concentration of this antigen in the sera of patients with thyroid diseases by inhibition assay. In sera from patients with destructive thyrotoxicosis, the concentration of the antigen was lower than that in sera from normal subjects and from Graves' disease patients. There was no significant difference between normal subjects and Graves' disease patients. The data suggest that measurement of the antigen in sera from patients with thyroid disease can be useful for the differential diagnosis of thyrotoxicosis.