The T cell Ag receptor (CD3/Ti) and the sheep E receptor (CD2) expressed on the surface of human T cells are both capable of initiating intracellular signals necessary for T cell activation. CD3/Ti interacts with Ag to initiate cellular immune responses. Although the exact function of CD2 is unknown, lymphocyte function-associated Ag 3 (LFA-3), a 55- to 70-kDa receptor expressed on a broad spectrum of hemopoietic and nonhemopoietic cells, has recently been shown to be its natural ligand. We show here that although purified multimeric LFA-3 is not capable of initiating transmembrane signaling events on its own, the combination of LFA-3 and the anti-CD2 mAb CD2.1 induces intracellular calcium increases, phosphatidylinositol second messenger generation and lymphokine secretion in the T cell leukemic line Jurkat. In order to study the signaling requirements of CD2, we compared the ability of CD2 mAb and LFA-3 to initiate activation signals in Jurkat and in three Jurkat-derived mutants. A CD3-CD2+ mutant failed to increase calcium or exhibit phosphatidylinositol hydrolysis to either the combination of agonist CD2 mAb 9-1 and 9.6 or LFA-3 and CD2.1. Reconstitution of the Ag receptor by transfection of the Ti-beta-chain restored the expression of the CD3/Ti complex and the ability to respond to either combination of CD2 ligands. However, no response to CD2 ligands was detected in a CD3+CD2+ mutant selected for signaling defects to CD3/Ti ligands. Complementation of the CD3/Ti signaling defect by cell fusion also restored competency to respond to CD2 agonists. These results demonstrate that LFA-3 under appropriate conditions can activate T cells via the CD2 complex and that this activation requires not only the cell surface expression of the CD3/Ti complex but also a functional Ag receptor pathway.