The pathophysiological role of thyroid blocking antibody (TBAb) in patients with adult primary hypothyroidism and the mechanism of TBAb action were studied. A sensitive bioassay for TBAb, which inhibits the TSH-induced cAMP accumulation, was established using normal human thyroid cells in culture. Thirty-four patients with primary hypothyroidism consisting of 17 goitrous and 17 non-goitrous patients were examined. Two out of 17 goitrous patients (11.8%) and three out of 17 non-goitrous patients (17.6%) were TBAb positive. There were no significant differences between TBAb positive and negative patients in terms of the severity of hypothyroidism or the titers of MCHA or TGHA. Four out of the five TBAb-positive IgGs had strongly positive thyrotropin binding inhibitor immunoglobulin activities. All five TBAb-positive IgGs inhibited the cAMP increase induced by Graves' IgG, but did not affect the action of either prostaglandin E1 or cholera toxin. However, three TBAb positive IgG also inhibited the cAMP increase induced by forskolin. These findings indicate: 1) TBAb is present in hypothyroid patients with autoimmune thyroiditis and TBAb may play a role in the pathophysiology of these patients. 2) TBAb may inhibit the action of TSH not only at the level of the TSH receptor, but also at a different site from the TSH receptor.