The cytoplasmic regions of the CD3 complex are presumably involved in signal transduction following ligand—receptor binding. We investigated the effects of incubating either stearic or oleic acid on the association of murine lymphocyte CD3 complex with the cytoskeleton. Both cytochalasin D, an inhibitor of microfilament formation, and W7, an inhibitor of calmodulin, inhibited capping of CD3. The association of CD3 with the cytoskeleton was confirmed by confocal laser scanning microscopy studies, which showed co-localization of the cross-linked CD3 receptors and the membrane attachment proteins ankyrin and fodrin. Although exogenous oleic acid increased plasma membrane fluidity, neither expression nor capping of CD3 receptors was increased. Nonetheless, oleic acid did increase uptake of tritiated thymidine after binding of anti-CD3 antibodies. Lymphoproliferation was progressively inhibited by both cytochalasin D and W7, confirming the importance of intact cytoskeleton for cellular activation.