We have used real-time video microscopy to study the mechanisms of platelet adhesion to type I collagen fibrils of distinct structure exposed to flowing blood. Electron microscopy analysis by surface replication demonstrated morphological differences between acid-insoluble fibrils, displaying a regularly repeating striated pattern (banded collagen), and acid-soluble fibrils generated by pepsin treatment of insoluble collagen, smaller in size with a helical configuration (nonbanded collagen). These structural differences proved to be related to the role of platelet integrin ₂β₁ in stabilizing adhesion to collagen under a variety of flow conditions. Blocking ₂β₁ function with a monoclonal antibody had no effect on platelet adhesion to insoluble type I collagen coated at high density on a glass surface, whereas there was an absolute dependence of ₂β₁ function for the initial permanent arrest of platelets and subsequent thrombus formation on pepsin-solubilized type I collagen under the same conditions. In contrast, reconstituted, banded fibrils prepared from pepsin-solubilized type I collagen supported platelet adhesion and thrombus development even when platelet ₂β₁ function was blocked, a process that was greatly accelerated by pre-exposure of this substrate to autologous plasma under flow. These results implicate a collagen receptor(s) on platelets other than ₂β₁ that can selectively engage domains in banded, but not nonbanded type I collagen when ₂β₁ function is blocked. In addition, collagen structure may regulate the extent and affinity of the binding under flow of plasma components such as von Willebrand factor and/or other _IIbβ₃ ligands.