There are three major classes of human Fcgamma receptors (FcgammaRI, FcgammaRII, and FcgammaRIII) and various isoforms of each class are capable of mediating phagocytosis. FcgammaRIIA is an unusual Fcgamma receptor in that it transmits a phagocytic signal in the absence of an additional receptor subunit. The cytoplasmic domain of FcgammaRIIA contains a conserved motif containing two copies of the sequence YXXL. The tyrosines (Y) within the motif are phosphorylated after receptor crosslinking and the integrity of these conserved sequences is required for efficient phagocytosis. The FcgammaRIIB receptors, FcgammaRIIB1 and FcgammaRIIB2, contain one copy of the cytoplasmic YXXL sequence and do not transmit a phagocytic signal. In B cells, FcgammaRIIB negatively regulates B-cell activation by the B-cell antigen receptor. Human macrophages express both FcgammaRIIA and FcgammaRIIB and while FcgammaRIIA mediates phagocytosis, the function of FcgammaRIIB in these cells is unknown. Using the epithelial/fibroblast-like cell line COS-1 as a model to examine the molecular events that regulate the phagocytosis of IgG-coated cells (EA), we investigated the effect of FcgammaRIIB on FcgammaRIIA signaling. FcgammaRIIB inhibited phagocytosis mediated both by FcgammaRIIA and by a chimeric FcgammaRIIA receptor containing the extracellular domain of FcgammaRI and the transmembrane and cytoplasmic domains of FcgammaRIIA. This inhibition occurred at an early signaling stage because tyrosine phosphorylation of the FcgammaRIIA cytoplasmic domain was inhibited after concurrent stimulation of these receptors with EA. FcgammaRIIB mutations showed the importance of the FcgammaRIIB YXXL for inhibition of FcgammaRIIA-mediated phagocytosis. Deletion of the FcgammaRIIB YXXL or conservative replacement of the YXXL tyrosine substantially reduced the inhibitory signal. FcgammaRIIB had a lesser inhibitory effect on phagocytosis by the Fcgamma receptor FcgammaRIIIA, which requires a gamma subunit to mediate a phagocytic signal. These results show that FcgammaRIIB negatively regulates phagocytic signaling by FcgammaRIIA and suggests that FcgammaRIIB plays a role in modulating FcgammaRIIA function in vivo.