RADIOTHERAPY IS USED IN THE MANAGEMENT OF PANCREATIC CANCER BECAUSE OF ITS HIGH PROPENSITY FOR LOCOREGIONAL RELAPSE: one third of patients succumb to localized disease. Thus, strategies to improve the efficacy of radiotherapy in pancreatic cancer are important to pursue. We used naturally serum-free, selectively permeable basement membranes and confocal microscopy of fluorescent antibody-stained human Panc-1, MiaPaCa-2, and BxPC-3 pancreatic cancer cell lines to investigate the effects of ionizing radiation on α(5)β(1) integrin fibronectin receptor expression and on α(5)β(1)-mediated invasion. We report that radiation rapidly induces pancreatic cancer cell invasion, and that radiation-induced invasion is caused by up-regulation of α(5)β(1) integrin fibronectin receptors by transcriptional and/or postendocytic recycling mechanisms. We also report that radiation causes α(5)β(1) up-regulation in Panc-1, MiaPaCa-2, and BxPC-3 tumor xenografts and that upregulated α(5)β(1) colocalizes with upregulated early or late endosomes in Panc-1 or BxPC-3 tumors, respectively, although it may colocalize significantly with both endosome types in MiaPaCa-2 tumors. Our results suggest that systemic inhibition of α(5)β(1)-mediated invasion might be an effective way to reduce radiation-induced pancreatic cancer cell invasion, thereby improving the efficacy of radiotherapy.