We have investigated the role of the cytoplasmic domains of LFA-1 in binding to ICAM-1 and in postadhesion events. Various truncated and chimeric forms of LFA-1 alpha (CD11a) and beta (CD18) chains were generated and transfected into murine fibroblast TNR-2 cells. Transfected fibroblasts expressing wild-type LFA-1 adhered only weakly to ICAM-1 immobilized on plastic, and phorbol ester pretreatment enhanced this adhesion significantly. In contrast, transfected cells expressing LFA-1 lacking both the alpha and the beta cytoplasmic domains, the beta cytoplasmic domain alone, or GPI-anchored LFA-1 adhered to immobilized ICAM-1 without prior activation. Truncation of the alpha cytoplasmic domain alone resulted in much reduced cell adhesion which could be only weakly upregulated by PMA. The presence of manganese dramatically enhanced the binding to ICAM-1 of LFA-1 lacking the alpha cytoplasmic domain or both cytoplasmic domains, whereas it had relatively little effect on wild-type LFA-1 or the mutant lacking the beta cytoplasmic domain. Soluble LFA-1, generated by phosphatidylinositol-specific phospholipase-C treatment of GPI-anchored LFA-1, was capable of binding ICAM-1+ cells. Although doubly truncated or GPI-anchored LFA-1 mediated cell adhesion to immobilized ICAM-1, cells expressing these mutants, as well as those expressing individual alpha and beta chain truncations, failed to spread out following this adhesion, whereas the wild-type transfectants did so readily. Manganese had no effect on cell spreading. Fluorescent staining of these cells indicated no significant variation in the distribution of LFA-1 on the cell surface. From these results we conclude that (1) cells expressing LFA-1 lacking both the alpha and the beta cytoplasmic domains adhere to ICAM-1 without prior stimulation, indicating the importance of LFA-1 cytoplasmic domains in inside-out signaling, (2) truncation of the alpha cytoplasmic domain alone inhibits cell adhesion by making LFA-1 nonresponsive to inside-out signaling, and (3) both cytoplasmic domains are required for cell spreading following adhesion to immobilized ICAM-1.