Antithymocyte globulin (ATG) is employed for the treatment and prevention of acute organ rejection after transplantation. However, the mechanisms underlying its immunomodulatory capacities beyond cellular depletion remains ill defined. A stable interaction between T-cells and professional antigen-presenting cells (APC) and full T-cell stimulation requires a complex molecular rearrangement at the T-cell/APC interface, the so called immunological synapse. Here we investigated, whether ATG affects T-cell/APC interactions. ATG concentration and time-dependently inhibited relocalization of the T-cell receptor/CD3 complex as well as adhesion molecules and cytoskeletal proteins of human peripheral blood T-cells and a human T-cell line towards the APC contact site. Moreover, ATG-treated peripheral blood T-cells were incapable to form conjugates with APCs. In conclusion, ATG impairs T-cell/APC conjugate formation, a mechanism that may help to understand the functional inactivation of peripheral blood T-cells that have escaped cellular depletion after ATG treatment.