This study examined Smad2- and Smad3-dependent transcription in 12 human head and neck squamous cell carcinoma (HNSCC) cell lines following treatment with transforming growth factor-beta1 (TGF-beta1). A markedly elevated level of TGF-beta1-induced Smad3 signalling was observed in one cell line (H357), whilst four cell lines (BICR31, H314, BICR56, BICR19) demonstrated absence of Smad3-dependent transcription that correlated with loss of TGF-beta1 growth inhibition; TGF-beta1-induced Smad2-dependent transcription was retained in two of these cell lines (H314, BICR31). Using transient expression of TGF-beta signalling components and a Smad3-dependent reporter assay, we show that BICR31 and H314 had defects of Smad4, BICR56 had abnormal TbetaR-II and BICR19 overexpressed Smad7. The results demonstrate that deregulated TGF-beta1-induced Smad signalling is common in HNSCC cell lines and can occur as a result of a variety of defects in the TGF-beta signal transduction pathway.