Stimulation of the TCR/CD3 complex on T cells initiates rearrangement of the actin cytoskeleton. The results presented show that a temporal increase in the appearance of filamentous actin begins immediately after stimulation of T cells with immobilized anti-CD3 mAb. The formation of filamentous actin in these stimulated cells reaches a steady state within 30 min after anti-CD3 mAb stimulation. At this time, pseudopod formation is observed and becomes progressively more evident over the next several hours. Experiments were done to investigate the role of the actin cytoskeletal associated proteins, alpha-actinin, vinculin, and talin, in the assembly of the actin cytoskeleton in anti-CD3 mAb-stimulated T cells. Using immunofluorescence, these three proteins are detected throughout the cytosol in resting T cells. However, after anti-CD3 mAb stimulation of the T cells, these proteins move to one pole of the cell. Electrophoresis followed by immunoblotting of T cell lysates prepared from resting as well as anti-CD3 mAb-stimulated cells revealed that alpha-actinin, but not vinculin or talin, was modified as a consequence of cell activation. Results show that alpha-actinin exists as a 105-kDa subunit in resting T cells, but that anti-CD3 mAb stimulation of T cells leads to the appearance of an 80-kDa lower molecular form of alpha-actinin. Experiments show that this occurs as a consequence of the 105-kDa subunit being proteolytically cleaved by calpain.