Fcγ receptors (FcγRs) play an essential role in the regulation of immune response due to their ability to bind immune complexes. Activating FcγRs may facilitate antigen presentation and dendritic-cell maturation, while in the late phase of the immune response, the inhibitory FcγRIIb may down-regulate B-cell activation upon cross-linking with activating receptors. In this study, we investigated the in vivo role of FcγRs on the modulation of humoral immune response. In order to get well-defined immune complexes that can bind to both the activating and the inhibitory FcγRs, we designed a mono-biotinylated single-chain fragment variable construct from the rat anti-mouse CD16/32 clone 2.4G2, linked to avidin–FITC, and tested its effect on the FITC–hapten-specific T-independent type 2 (TI-2) and T-dependent (TD) immune response. When injected intravenously in mice, the complex bound to a small portion of B220+, CD11bhigh and CD11chigh cells and was localized in the spleen on marginal zone macrophages 15 min after treatment. When applied as a booster following primary immunization with TI-2 (FITC–dextran) or TD (FITC–keyhole limpet haemocyanin) antigens, the complex elevated the number of hapten-specific IgM/IgG-producing B cells. This effect was diminished in CD16KO mice, suggesting that the activating-type FcγRIII might be a key mediator of this mechanism.