This study demonstrates the variable expression of ICAM-1 and leukocyte function antigen-3 (LFA-3) on four tumor cell lines (COLO526, K562, Daudi, and HT-29). In addition, phorbol ester (PMA) activation of lymphocytes modulated LFA-1 from a uniform to a clustered surface distribution; whereas after treatment with high levels of Mg2+ ions, the unique epitope for high-affinity LFA-1 was identified using clone Mab24. Using a flow cytometric adhesion assay it was demonstrated that PMA-activated lymphocytes formed conjugates with COLO526 and Daudi, and that these conjugates were inhibited by anti-CD2 with varying inhibition by LFA-1 clones MHM24 and 25.3.1. When lymphocytes were induced to express the high-affinity form of LFA-1, conjugates were identified with COLO526, K562, and Daudi and these conjugates were sensitive to the presence of both CD2 and LFA-1 antibodies. Further studies using confocal microscopy confirmed significant adhesion between peripheral blood lymphocytes pretreated with either PMA or high levels of Mg2+ and the adherent cell line COLO526. In conclusion, this unique study has demonstrated for the first time the important role of the active form of LFA-1 on the lymphocyte cell surface for conjugate formation with an ICAM-1-expressing tumor cell; also, two pathways of cell signaling were identified for conjugate formation to occur.