Studies on frequency and distribution pattern of TSH receptor (TSHR) and Gs alpha protein (gsp) mutations in toxic thyroid nodules (TTNs) reported conflicting results, most likely also related to the different screening methods applied and the investigation of only part of exon 10 of the TSHR. Therefore, we screened a consecutive series of 31 TTNs for both TSHR and gsp mutations by direct sequencing of exon 9 and the entire exon 10 of the TSHR gene and exons 7-10 of the gsp gene. Somatic TSHR mutations were identified in 15 of 31 TTNs. TSHR mutations were localized in the third intracellular loop (Asp619Gly and Ala623Val), the sixth transmembrane segment (Phe631Leu and Thr632Ile, Asp633Glu) and the second extracellular loop (Ile568Thr). One mutation was found in the extracellular TSHR domain (Ser281Asn). Two new TSHR mutations were identified. One involves codon 656 in the third extracellular loop (Val656Phe). The other new mutation is a 27-bp deletion in the third intracellular loop resulting in deletion of 9 amino acids at codons 613-621. Transient expression of the new TSHR mutations in COS-7 cells demonstrated their constitutive activity. No mutation was found in exons 7-10 of the gsp gene. This finding was confirmed by an allele-specific PCR for mutations in gsp codons 201 (Arg-->His, Cys) and 227 (Gln-->His, Arg). Our data indicate that constitutively activating TSHR mutations can be found in 48% of TTNs and thus currently represent the most frequent molecular mechanism known in the etiopathogenesis of TTNs. Moreover, the absence of gsp mutations in our series argues for an only minor role of these mutations in TTNs. Constitutive activation of the TSHR by a deletion in a region that might be involved in G protein coupling of the TSHR offers new insights into TSHR activation.