Lymphocytes activate adhesion to intracellular adhesion mlecule 1 (ICAM-1) via leukocyte function associated antigen 1 (LFA-1), their major beta 2 integrin, in response to PMA (phorbol 12-myristate 13-acetate) without an increase in the number of receptors expressed. The molecular details of the mechanism are unknown. To determine the effect of PMA activation on LFA-1 movement within the plasma membrane, we used the single particle tracking technique to measure the diffusion rate of LFA-1 molecules on EBV-transformed B cells before and after PMA activation. Diffusion of LFA-1 on unactivated cells was restricted compared to CR1 (CD35), another transmembrane protein of equivalent size. PMA caused a 10-fold increase in the diffusion rate of LFA-1 without any effect on CD35. The increased LFA-1 motion induced by PMA was random, not directed, indicating that it was due to a release of constraints rather than the application of forces. The diffusion rates of LFA-1 are consistent with cytoskeletal attachment before and free diffusion after PMA. Cytochalasin D led to an equivalent increase in mobility and, at low doses, stimulated adhesion, implying that the nonadhesive state of LFA-1 is actively maintained by the lymphocyte cytoskeleton.