Integrin-mediated adhesion to extracellular matrix proteins confers resistance to radiation-or drug-induced genotoxic injury. To analyse the underlying mechanisms specific for β1-integrins, wild-type β1A-integrin-expressing GD25β1A cells were compared to GD25β1B cells, which express signaling-incompetent β1B variants. Cells grown on fibronectin, collagen-III, β1-integrin-IgG or poly-l-lysine were exposed to 0–6 Gy X-rays in presence or depletion of growth factors and phosphatidylinositol-3 kinase (PI3K) inhibitors (LY294002, wortmannin). In order to test the relevance of these findings in tumor cells, human A-172 glioma cells were examined under the same conditions after siRNA-mediated silencing of β1-integrins. We found that β1A-integrin-mediated adhesion to fibronectin, collagen-III or β1-IgG was essential for cell survival after radiation-induced genotoxic injury. Mediated by PI3K, pro-survival β1A-integrin/Akt signaling was critically involved in this process. Additionally, the β1-integrin downstream targets p130Cas and paxillin-impaired survival-regulating PI3K-dependent JNK. In A-172 glioma cells, β1-integrin knockdown and PI3K inhibition confirmed the central role of β1-integrins in Akt-and p130Cas/paxillin-mediated prosurvival signaling. These findings suggest β1-integrins as critical regulators of cell survival after radiation-induced genotoxic injury. Elucidation of the molecular circuitry of prosurvival β1-integrin-mediated signaling in tumor cells may promote the development of innovative molecular-targeted therapeutic antitumor strategies.