In contrast to nonneoplastic keratinocytes, human squamous carcinoma cell lines are able to proliferate in the presence of transforming growth factor-beta (TGF-beta) in vitro. This has raised the question whether, how frequently, by which mechanism, and at which stage of development squamous carcinomas escape from TGF-beta control in vivo. We have developed a method to rapidly identify the most common molecular alterations in the TGF-beta signaling pathway by combining measurements of the levels and the activation state of Smad signaling intermediates with DNA-based diagnostic assays. In this report, we demonstrate the validity of this approach using a panel of seven squamous cell carcinoma (SCC) lines known to be refractory to TGF-beta-mediated cell cycle arrest. Each of the SCCs expressed the pathway-restricted Smad proteins, Smad2 and-3. Furthermore, treatment with TGF-beta induced phosphorylation of Smad2 in each of the SCCs with the exception of the two cell lines that carry inactivating mutations of the TGF-beta type II receptor. Three of the remaining SCC lines failed to express the common mediator Smad4, two on the basis of loss of transcription and one by a posttranscriptional mechanism. Thus, a mechanism for TGF-beta resistance was identified in five of the seven tumor cell lines. Interestingly, in the two remaining lines, no abnormalities of signaling intermediates were found, and TGF-beta was able to activate TGF-beta-responsive promoters. This suggests that the ability of these two cell lines to grow in the presence of TGF-beta is due to factors extraneous to the TGF-beta pathway itself. Application of our protein-based strategy to interrogate the TGF-beta signaling pathway should allow us to determine whether or not and, if so, how and at which stage human squamous cell carcinomas become TGF-beta resistant in vivo.