Abstract The high-affinity IgG receptor, FcγRI (CD64), is constitutively expressed exclusively on professional APCs. Human FcγRI binds monomeric IgG with high affinity and is, therefore, saturated in vivo. The binding of IgG to FcγRI causes receptor recycling, while Abs that cross-link FcγRI cause rapid down-modulation of surface FcγRI. Because studies performed in the absence of ligand may not be representative of FcγRI modulation in vivo, we investigated the ability of FcγRI-cross-linking Abs and non-cross-linking derivatives to modulate FcγRI in the presence and absence of ligand. In the absence of ligand mAb H22 and wH22xeGFP, an enhanced green fluorescent protein (eGFP)-labeled fusion protein of H22, cross-linked and rapidly down-modulated surface FcγRI on the human myeloid cell line, U937, and its high FcγRI-expressing subclone, 10.6. This effect was dependent on the concentration of fusion protein and the level of FcγRI expression and correlated with internalization of both wH22xeGFP and FcγRI, itself, as assessed by confocal microscopy. A single-chain Fv version, sFv22xeGFP, which does not cross-link FcγRI, was unable to modulate FcγRI in the absence of IgG. However, if ligand was present, treatment with either monovalent or cross-linking fusion protein led to intracellular receptor accumulation. These findings suggest at least two alternate mechanisms of internalization that are influenced by ligand and demonstrate the physiologic potential of FcγRI to transport a large antigenic load into APCs for processing. These studies may lead to the development of better FcγRI-targeted vaccines, as well as therapies to down-modulate FcR involved in autoimmune diseases.