Intense investigation has centered on understanding the regulation of integrin cell adhesion receptors. In the present study, we propose that variant N-glycosylation represents an important mechanism for regulation of β1, but not β3 or β5 integrins. We find that expression of oncogenic ras in HD3 colonocytes causes increased α2-6 sialylation of β1 integrins, whereas expression of dominant-negative ras induces decreased α2-6 sialylation, relative to cells with wild-type ras. In contrast, neither β3 nor β5 integrins are α2-6 sialylated, regardless of the state of ras activation. Results from RT-PCR analyses suggest that differential integrin sialylation is due to a ras-dependent alteration in the expression of ST6Gal I, the enzyme that adds α2-6-linked sialic acids. Cells that express differentially sialylated β1 integrins exhibit altered adhesion to collagen I (a β1 ligand), but not to vitronectin (a β3 or β5 ligand). Similarly, the enzymatic removal of cell surface sialic acids from control cells alters binding to collagen, but not to vitronectin. Finally, using a cell-free receptor/ligand-binding assay, we show that purified, desialylated α1β1 integrins have diminished collagen-binding capability, providing strong evidence that sialic acids play a causal role in regulating β1 integrin function.