We investigated the role of acetylated microtubules in the antigen-specific interaction of T helper and antigen-presenting cells (APCs). In T cells, acetylated microtubules concentrated at contact site with APCs, surrounding clusters of CD3 and LFA-1. TcR engagement induced a transient deacetylation of microtubules at early times and an enhanced acetylation at late times. Confocal videomicroscopy studies revealed that the HDAC6 tubulin deacetylase was translocated and concentrated at the contact site of T cells with APCs. Overexpression of HDAC6 but not a dead deacetylase mutant in T cells disorganized CD3 and LFA-1 at the immune synapse. This effect was reverted by treatment with the deacetylase inhibitor trichostatin A. The antigen-specific translocation of the microtubule organizing center (MTOC) and IL-2 production were also severely impaired by overexpression of HDAC6. Our results underscore the key role for HDAC6 in the organization of the immune synapse and the antigen-specific reorientation of the MTOC.