T cell activation occurs when the T cell receptor (TCR) is engaged by an antigen-MHC complex on the surface of an antigen-presenting cell (APC). Additional signals provided by accessory molecules serve to modulate this response. Independent of TCR engagement, treatment of T lymphocytes with a combination of phorbol ester and CD28 ligation will result in a proliferative response. This also induces homotypic adhesion mediated by LFA-l/ICAM interactions. We demonstrate that the prevention of homotypic interactions between T cells resulted in a two- to fivefold increase in the proliferative response. This occurred whether the homotypic interactions were prevented by blockade of LFA-1, by the use of plate immobilized antibodies against other cell surface molecules, or by culture at low cell density. We further demonstrate that the increased proliferation was a result of interference with a negative signal delivered to the T cell as a result of ICAM-3-mediated events. These data demonstrate LFA-1/ICAM-3 interactions between T cells in turn regulate an LFA-1-independent pathway that results in homotypic adhesion and a downregulation of the proliferative response of activated T cells.