We have characterized the immunobiology of the interaction of intercellular adhesion molecule 3 (ICAM-3; CD50) with its counter-receptor, leukocyte function-associated antigen 1 (LFA-1; CD11a/CD18). Purified ICAM-3 supported LFA-1-dependent adhesion in a temperature- and cation-dependent manner. Activation of cells bearing LFA-1 increased adhesiveness for ICAM-3 in parallel to adhesiveness for ICAM-1. Although CBR-IC3/1 monoclonal antibody (mAb) blocked adhesion of cells to purified LFA-1, when tested alone, neither CBR-IC3/1 nor five novel ICAM-3 mAbs characterized here blocked adhesion of cells to purified ICAM-3 or homotypic adhesion. Two ICAM-3 mAbs, CBR-IC3/1 and CBR-IC3/2, were required to block LFA-1-dependent adhesion to purified ICAM-3- or LFA-1-dependent, ICAM-1-, ICAM-2-independent homotypic adhesion of lymphoid cell lines. Two ICAM-3 mAbs, CBR-IC3/1 and CBR-IC3/6, induced LFA-1-independent aggregation that was temperature and divalent cation dependent and was completely inhibited by ICAM-3 mAb, CBR-IC3/2, recognizing a distinct epitope. Purified ICAM-3 provided a costimulatory signal for proliferation of resting T lymphocytes. mAb to ICAM-3, together with mAbs to ICAM-1 and ICAM-2, inhibited peripheral blood lymphocyte proliferation in response to phytohemagglutinin, allogeneic stimulator cells, and specific antigen. Inhibition was almost complete and to the same level as with mAb to LFA-1, suggesting the most functionally important, and possibly all, of the ligands for LFA-1 have been defined.