TSH receptor stimulating antibodies (TSAb) are well known in the pathogenesis of Graves' disease. Recently mutations in the DNA coding for the TSH-receptor (TSHR) have been revealed in autonomously functioning thyroid nodules (AFTN). In this study we looked for mutations in 9 patients with AFTN. DNA was extracted separately from the nodules and the paranodular thyroid tissues. A fragment of the TSHR gene (bp 1762-1976) was amplified by PCR. The amplified DNA was digested with Hph I, Taq I, Kpn I and Eco RI, respectively, and the resulting fragments were analyzed by gel electrophoresis. Mutations were detected in the nodules of 3 of the 9 patients. In the nodules of 2 patients we found simultaneous mutations affecting the restriction sites of Hph I and Taq I. In the 3rd patient a mutation affecting the restriction site of Hph I was observed. The simultaneous occurrance of TSHR-mutations altering Hph I (Position 631) and Taq I (position 632) restriction sites in AFTN have not been described so far.