Both Fc gamma receptors on human neutrophils (Fc gamma RIIa and Fc gamma RIIIb) are capable of initiating signal transduction after multivalent cross-linking. However, immune complexes most likely activate neutrophils by a combined homotypic and heterotypic cross-linking of Fc gamma Rs. We have investigated the effect of homotypic and heterotypic Fc gamma R cluster formation on changes in the intracellular free Ca2+ concentration. Combined heterotypic and homotypic cluster formation resulted in a Ca2+ response that was strongly enhanced as compared to the sum of both individual Fc gamma R responses. This synergistic response was caused by the formation of heterotypic clusters of Fc gamma Rs and not by the simultaneous formation of homotypic clusters. This conclusion was supported by experiments with a bispecific antibody binding to both Fc gamma RIIa and Fc gamma RIIIb. The heterotypic Fc gamma R cross-linking results in efficient activation of Ca2+ influx, probably caused by a more pronounced depletion of intracellular Ca2+ stores. Stimulation with immune complexes also induced Ca2+ influx in normal neutrophils, but not in Fc gamma RIIIb-deficient neutrophils. The synergism between both Fc gamma Rs was also apparent in other responses of neutrophils, such as the activation of the respiratory burst. This study shows that the two different Fc gamma Rs on neutrophils complement each other in mediating an important cellular response.