OBJECTIVE: It is claimed that second generation thyrotropin-binding inhibitory immunoglobulin (TBII) assays have a very high sensitivity for the diagnosis of Graves' hyperthyroidism (GH). However, studies evaluating the accuracy of TBII have been retrospective in nature and/or GH had not been diagnosed independently of TBII. The aim of the present study, therefore, was to prospectively evaluate the frequency and characteristics of TBII-seronegative patients in a population of untreated GH diagnosed independent of serum TBII. DESIGN: Prospective multicentre observational study. PATIENTS: A total of 259 consecutive untreated patients with a first episode of GH, diagnosed independent of serum TBII. TBII levels were measured by second generation assay and correlated to thyroid function, clinical characteristics and exposure to environmental factors. RESULTS: Serum TBII was positive in 245 (94.6%) patients and negative (< 2 IU/l) in 14 (5.4%) patients. TBII-seronegative patients had lower fT4 (median 42.5 vs. 53.9 pmol/l, P = 0.02), T3 (median 3.55 vs. 4.90 nmol/l, P < 0.01) and fT3-index (median 4.30 vs. 6.27, P < 0.01) compared to TBII-seropositive patients. None of the TBII-seronegative patients had TSH-receptor activating mutations, Graves' orbitopathy or pretibial myxedema. Serum TBII was positively correlated to free T3 (fT3)-index and free T4 (fT4)-index (P < 0.01), goitre size (P < 0.01) and the prevalence of Graves' orbitopathy (P < 0.01). There were no significant differences between TBII-seropositive and TBII-seronegative patients in environmental factors. CONCLUSION: The prevalence of TBII-seronegativity in untreated patients with GH is 5.4% using a second generation assay. TBII-seronegative patients have biochemically less severe thyrotoxicosis and no Graves' orbitopathy. TBII-seronegative and TBII-seropositive patients apparently belong to the same population of GH, albeit the severity of the autoimmune attack is less in TBII-seronegative patients.