Recently, we demonstrated that S100C/A11 comprises an essential pathway for growth suppression by TGFbeta in normal human keratinocytes. Nuclear transfer of S100C/A11 was a hallmark of the activation of the process. In the present study, we examined the possible deterioration in the pathway in human squamous cancer cell lines, focusing on intracellular localization of S100C/A11 and its functional partners Smad3 and Smad4. All four human squamous cancer cell lines examined (A431, BSCC-93, DJM-1, and HSC-5) were resistant to growth suppression by TGFbeta. In BSCC-93, DJM-1, and HSC-5 cells exposed to TGFbeta, S100C/A11 was not transferred to the nuclei, and p21(WAF1) was not induced. Overexpression of nucleus-targeted S100C/A11 partially recovered induction of p21(WAF1) and p15(INK4B) and growth suppression by TGFbeta1 in these cells. These results indicate that the deterioration in the S100C/A11-mediated pathway conferred upon the cancer cell lines resistance to TGFbeta. In A431 cells, S100C/A11, Smad3, and Smad4 were simultaneously transferred to the nuclei, and p21(WAF1) was induced upon exposure to TGFbeta. We provide evidence to indicate that refractoriness of A431 cells to TGFbeta was probably because the amount of p21(WAF1) induced by TGFbeta was insufficient to counteract cyclin A, which is highly overexpressed in A431 cells. Thus, the newly found S100C/A11-mediated pathway is at least partly involved in conferring upon human squamous cell cancers resistant to TGFbeta-induced growth suppression, which is considered to play a critical role for the initiation and progression of many human cancers.