Sensitivity to transforming growth factor-beta is impaired in thyroid tumours. Similar to Mad -- Mother Against Decapentaplegic-(Smad)4 is frequently altered in cancers, but its involvement in this system is unknown. We analysed 56 thyroid tumours of various histotypes for Smad4 mutations by PCR-SSCP and sequencing, linking them to Smad4 reactivity as examined by immunohistochemistry (IHC), and 29 of them also for abnormalities in RNA expression due to alternative splicing. In all, 15/56 cases (27%), both benign and malignant lesions, harbour alterations of Smad4 coding sequence. We found several novel intragenic mutations (13 missense, two silent, one frameshift and one large insertion-deletion), with high incidence in the linker region. A subset of mutated tumours failed to express Smad4 protein by IHC. We have also detected four alternatively spliced tumour-associated Smad4 isoforms, lacking portions of the linker region, and three more due to unreported internal exon-exon rearrangements. Smad4 is both frequently mutated and deregulated by aberrant splicing in thyroid tumours and these alterations may contribute as an early event to thyroid tumorigenesis.