The α₂β₁ integrin is a major collagen receptor on platelets. Although it has been proposed that α₂β₁, like α_IIbβ₃, undergoes agonist-induced activation, neither the potential contributions of α₂β₁ receptor/ligand internalization to the increase in ligand binding nor the roles of the α₂ and β₁ cytoplasmic domains in activation of this integrin have been previously explored. Activation of α₂β₁ was assessed with fluorescein isothiocyanate–labeled soluble type I collagen binding to platelets by flow cytometry. Although collagen internalization in response to agonist activation of platelets was significant, agonist-induced collagen binding still occurred under conditions that block internalization, with minimal changes in cell surface α₂β₁ expression. Introduction of cell-permeable peptides containing the α₂ cytoplasmic tail, and especially the membrane proximal KLGFFKR domain, induced α₂β₁ activation in resting platelets, whereas a cell-permeable peptide containing the β₁ cytoplasmic tail was without effect. Thus, collagen binding to stimulated platelets is increased due to α₂β₁ activation, in addition to internalization, and the GFFKR motif appears to play an important role in the activation process.