B2A2-CD4-8- cells represent a rare subpopulation of thymocytes normally comprising 0.5% of the total adult thymus. These cells are known to express CD3-associated T cell receptor (TcR) alpha/beta molecules. In the present study we have examined the functional capacity of alpha/beta heterodimers on B2A2-CD4-8- cells. In the presence of monoclonal antibody (mAb) specific for either murine CD3 or TcR expressing the V beta 8-encoded beta chain (F23.1), B2A2-CD4-8- cells proliferated. Such proliferation was blocked by mAb to interleukin 2 receptor (IL 2R), suggesting an autocrine mechanism involving IL 2 production and subsequent utilization. IL 2 and also IL 3 production by mAb-stimulated B2A2-CD4-8- cells was directly confirmed. Furthermore, a panel of B2A2-CD4-8- clones were derived to assess the role of the TcR in cytolysis. Many clones were isolated which killed Fc receptor-bearing P815 target cells only in the presence of F23.1 mAb. Finally, in vivo treatment of neonatal mice with F23.1 mAb resulted in a marked reduction of V beta 8+ B2A2-CD4-8- thymocytes. Collectively, these results indicate that the TcR alpha/beta complex on CD4-CD8-B2A2- cells is fully capable of transducing signals that lead to proliferation, lymphokine production and cytolysis in vitro, as well as to disappearance of this subset from the thymus in vivo.