To determine the functional role of the two isoforms of Fc gamma RIII (CD16) (IIIA, IIIB), the signal transduction capabilities of wild-type and mutant forms of these receptors were analyzed in transfected lymphoid, myeloid, and fibroblastic cell lines. Functional reconstitution of receptor signalling was observed in hematopoietic T and mast cells, and was absent in nonhematopoietic (CHO) cells. Fc gamma RIIIA, a hetero-oligomeric receptor composed of a ligand-binding subunit alpha and dimeric gamma chains, generated both proximal and distal responses in Jurkat and P815 cells, typical of what is seen in natural killer cells and macrophages upon receptor activation. In contrast, Fc gamma RIIIB, which is normally attached to the cell surface via a glycosyl-phosphatidylinositol anchor, was incapable of transducing signals. After crosslinking, Fc gamma RIIIA signalling was dependent only upon the gamma chain. Fc gamma RIIIA chimeras in which the alpha subunit transmembrane and cytoplasmic domains were substituted with the corresponding gamma chain sequences functioned as well as wild-type hetero-oligomeric receptors. These data indicate that the ability of the Fc gamma RIIIA complex to activate the appropriate pathways for cell activation is cell-type restricted and independent of the transmembrane and cytoplasmic domains of the alpha subunit. The presence of the gamma chain is responsible for the assembly of, as well as the signal transduction by, the functional cell surface complex.