Previous studies have indicated that titers of thyroid-specific autoantibodies decrease in patients with autoimmune thyroid disease during antithyroid drug therapy. In keeping with these observations has been the accumulating evidence that such drugs may have an immunosuppressive action both in vivo and in vitro. To further analyze the mechanism of such activity, we have used an indirect hemolytic plaque assay based on protein A-coated sheep red blood cells and a specific plaqueforming cell (PFC) assay based on human thyroglobulin-linked sheep red blood cells. When freshly prepared peripheral mononuclear cells were exposed to increasing concentrations of methimazole or propylthiouracil (PTU), there was no significant effect on spontaneous plaque-forming cell responses (PFC). However, after incubation for more than 6 days with methimazole or PTU, there was a marked dose-related inhibition of pokeweed mitogen stimulated PFC, the degree of inhibition varying from one individual to another. overall, there were 31.5 ± 9.6% and 32.5 ± 5.2% reductions in plaque formation with 10-4 M methimazole or PTU, respectively, a level which probably approximates intrathyroidal thionamide concentrations in patients. A similar inhibitory response was seen in the human thyroglobulin-PFC assay using peripheral mononuclear cells from patients with autoimmune thyroiditis, confirming the potential inhibition of thyroid-specific autoantibody production by these compounds. Furthermore, at 10-4 M there was no effect of PTU or methimazole on cell survival, as assessed by trypan blue exclusion. These data suggested that the thionamide drugs interfered with immunoglobulin production, rather than secretion, at concentrations which were likely to be present intrathyroidally in many treated patients.