The present study investigated possible receptor-like characteristics of glycosylphosphatidylinositol (GPI)-linked antigens on human monocytes and granulocytes by measuring cytoplasmic calcium fluxes and the oxidative burst in cells following cross-linking of GPI-linked antigens. Cross-linking of cell-bound anti-CD14, -CDw52 and -CD55 induced cytoplasmic calcium fluxes and oxidative bursts in unprimed human monocytes similar to those observed following Fc gamma R cross-linking. In granulocytes primed with 200 mM N-formyl-Met-Leu-Phe (FMLP), cross-linking of cell-bound anti-CD16, -CD24, -CD59 and -CD67 led to calcium fluxes and activation of the oxidative burst. The oxidative bursts mediated by GPI-linked antigens were stronger than those induced by 200 nM FMLP, even though FMLP induced a larger increase in cytoplasmic calcium concentration. The responses were likely to be independent of Fc gamma R interactions as F(ab')2 fragments of IgG or IgM antibodies were used in the experiments. Activating effects of monoclonal antibody to GPI-linked antigens were not observed in cells from patients with paroxysmal nocturnal hemoglobinuria, which are deficient in GPI-linked antigens. In addition, treatment with GPI-specific phospholipase C led to inhibition of cell activation through GPI-linked antigens but not through transmembrane receptors. Cross-linking of a number of non-GPI-linked antigens (CD11a, CD18, CD31, CD35, CD43, and CD45) neither induced calcium fluxes, nor activated the oxidative burst. The results indicate that most, if not all, GPI-linked surface glycoproteins on myeloid cells are capable of mediating cell activation and suggest that the GPI anchor is a structure facilitating signal transduction.