The integrin VLA-2 (alpha 2 beta 1), generally considered to represent the specific collagen receptor on human endothelial cells, contains an alpha 2-subunit inserted I domain with structural similarity to the type A domains found within the recently described superfamily of receptor-ligand recognition proteins. This region of the cDNA has now been isolated and used for molecular and functional characterization of this heterodimeric receptor complex. Comparative sequence analysis with the porcine homologue revealed 93% amino acid sequence identity, suggestive of a developmentally conserved function. To complete structure/function studies, this region of the human cDNA was expressed as a chimeric protein in Escherichia coli, and a rabbit polyclonal antibody (anti-I domain) was used to study determinants of endothelial cell attachment and spreading in vitro. Quantifiable and visual disruption of endothelial cell attachment to gelatin, type I collagen, and laminin was evident using the specific anti-I domain antibody, with minimal inhibitory effects demonstrable using fibronectin or fibrinogen matrices. Therefore, these data would suggest that the alpha 2 beta 1 I domain confers ligand-binding specificity for both known alpha 2 beta 1 substrates (laminin and collagen), and that this region subserves a regulatory function in the molecular processes controlling endothelial cell attachment and spreading in vitro.