Loss of adherence to substrate can, by itself, induce apoptosis (anoikis) in epithelial cells, but does not do so in fibroblasts. To test the idea that adherence transmits signals that inhibit apoptosis even in fibroblasts, we took advantage of the greatly increased adherence to the substratum observed in NIH3T3 cell lines that overexpress thymosin beta four. We treated overexpressing (OE) and vector control lines with either ultraviolet light (UV) or tumor necrosis factor alpha (TNF alpha). When the cells were on a substratum, the more adherent OE cells were 2-fold more resistant to apoptosis induced by either treatment than vector controls. In contrast, when the cells were treated with either agent while in suspension, the difference in resistance between OE cells and vector controls was lost. Thus the increased resistance to apoptosis was dependent on adherence. There was no difference in the content of bcl-2 in the OE cells vs the controls. A connection between pp125FAK and resistance to apoptosis has been previously shown in primary cultures of fibroblasts. The Tbeta4 overexpressing cells have approximately 1.4x more pp125FAK than the controls, and the kinase is approximately 2-fold more phosphorylated in adherent OE cells than in the vector controls. The phosphorylation of pp125FAK decreased strikingly when the cells were put into suspension. In addition, twice as much paxillin associated with pp125FAK in OE adherent cells as in vector controls, but this difference was also lost in suspended cells. Our results support the concept of an adherence dependent pp125FAK-paxillin signalling pathway in fibroblasts that inhibits damage-induced apoptosis.