AbstractThe motility of T cells depends on the dynamic spatial regulation of integrin‐mediated adhesion and de‐adhesion. Cathepsin X, a cysteine protease, has been shown to regulate T‐cell migration by interaction with lymphocyte function associated antigen‐1 (LFA‐1). LFA‐1 adhesion to the ICAM‐1 is controlled by the association of actin‐binding proteins with the cytoplasmic tail of the β2 chain of LFA‐1. Cleavage by cathepsin X of the amino acid residues S769, E768 and A767 from the C‐terminal of the β2 cytoplasmic tail of LFA‐1 is shown to promote binding of the actin‐binding protein α‐actinin‐1. Furthermore, cathepsin X overexpression reduced LFA‐1 clustering and induced an intermediate affinity LFA‐1 conformation that is known to associate with α‐actinin‐1. Increased levels of intermediate affinity LFA‐1 resulted in augmented cell spreading due to reduced attachment of T cells to the ICAM‐1‐coated surface. Gradual cleavage of LFA‐1 by cathepsin X enables the transition between intermediate and high affinity LFA‐1, an event that is crucial for effective T‐cell migration.