Pancreatic cancer cells express 2 known collagen-binding integrins, alpha2beta1 and alpha1beta1. The ligand-binding specificity of alpha1beta1 and the integrin/s responsible for mediating the malignant phenotype on type I collagen in the 3-dimensional (3D) tumor microenvironment have not been determined in pancreatic cancer. The aim of the present study was to determine the ligand-binding specificities of the alpha2beta1 and alpha1beta1 integrins using a novel 3D in vitro model of pancreatic cancer.We used 3D type I collagen-glycosaminoglycan scaffolds in adhesion and proliferation assays with pancreatic cancer cell lines, as well as affinity chromatography and inhibition of adhesion assays.We demonstrate for the first time that CFPAC, BxPC-3, Colo-357, FG, and Panc-1 cells attach to 3D type I collagen scaffolds in an alpha2beta1-specific manner and that this integrin-specific adhesion is required for subsequent cell proliferation. MiaPaCa-2 cells, which do not express the alpha2beta1 or alpha1beta1 integrins, do not attach or proliferate on 3D type I collagen scaffolds. We also demonstrate the novel finding that the alpha1beta1 integrin is a type IV collagen receptor in pancreatic cancer cells.These data indicate that targeting alpha2beta1 integrin-specific type I collagen adhesion may have therapeutic value in the treatment of pancreatic cancer.