The authors have previously demonstrated that α2β1 integrin-mediated pancreatic cancer cell adhesion to Type I collagen is Mg2+-dependent, inhibited by Ca2+, and that this integrin, purified from cell lysates using Type I-collagen-sepharose in Mg2+, can be eluted with Ca2+. In the present study, the authors examined the divalent cation-dependency of α2β1 integrin-mediated pancreatic cancer cell adhesion, migration and proliferation on Type I collagen, an extracellular matrix protein shown to be highly up-regulated, and to promote the malignant phenotype in vitro and in vivo. The results indicate that cells attach to Type I collagen maximally when Mg2+ is greater than 1 mM, and that addition of increasing concentrations of Ca2+ reduces this adhesion. These effects are reversible, in that previous cell attachment in Mg2+ can be reversed by adding Ca2+, and vice versa. They also demonstrate that pancreatic cancer cells migrate and proliferate on Type I collagen in Mg2+ alone, but maximally when Mg2+ is present at concentrations that promote maximal cell adhesion and Ca2+ is present at concentrations less than Mg2+. Cell adhesion and proliferation assays, as well as affinity chromatography on Type I collagen using anti-integrin function-blocking monoclonal antibodies indicate that the effects of these divalent cation shifts are mediated specifically by the α2β1 integrin. As pancreatic juice contains over 1,200-fold more Mg2+ than Ca2+ and solid tumors are characterized by increased magnesium load, these data indicate that such pathophysiological divalent cation shifts could be involved in the activation of the α2β1 integrin-mediated malignant phenotype on Type I collagen in the pancreatic cancer. © 2008 Wiley-Liss, Inc.