We have previously reported that the murine T cell line EL-4 has an aggregating phenotype, displaying homotypic aggregation (HTA) when exposed to monoclonal antibodies targeting specific cell surface molecules such as leukocyte function-associated antigen-I (LFA-1). We have used this property of EL-4 cells to isolate additional HTA-inducing MAb by screening a panel of hybridomas that were generated from rats immunized with EL-4 cells. We have isolated a novel anti-Thy-1 MAb (termed FF-10) that is a powerful inducer of HTA in EL-4 cells. In addition to induction of HTA, FF-10 also induces splenocyte proliferation but inhibits anti-CD3-driven T cell proliferation. Thy-1-induced HTA cannot be blocked with MAb targeting intercellular adhesion molecule-I and -2 (ICAM-1, ICAM-2) or LFA-1. Thus, the FF-10 MAb represents a novel and unique tool to investigate the diverse roles of the murine Thy-I molecule in T cell activation, proliferation and apoptosis.