A subset of integrin α subunits contain an I domain, which is important for ligand binding. We have deleted the I domain from the β2 integrin lymphocyte function-asssociated antigen-1 (LFA-1) and expressed the resulting non–I domain-containing integrin (ΔI-LFA-1) in an LFA-1-deficient T cell line. ΔI-LFA-1 showed no recognition of LFA-1 ligands, confirming the essential role of the I domain in ligand binding. Except for I domain monoclonal antibodies (mAbs), ΔI-LFA-1 was recognized by a panel of anti-LFA-1 mAbs similarly to wild-type LFA-1. However, ΔI-LFA-1 had enhanced expression of seven mAb epitopes that are associated with β2 integrin activation, suggesting that it exhibited an “active” conformation. In keeping with this characteristic, ΔI-LFA-1 induced constitutive activation of α4β1 and α5β1, suggesting intracellular signaling to these integrins. This “cross-talk” was not due to an effect on β1 integrin affinity. However, the enhanced activity was susceptible to inhibition by cytochalasin D, indicating a role for the cytoskeleton, and also correlated with clustering of β1 integrins. Thus, removal of the I domain from LFA-1 created an integrin with the hallmarks of a constitutively active receptor mediating signals into the cell. These findings suggest a key role for the I domain in controlling integrin activity.