The initiation of most cytotoxic immune responses requires MHC class I-restricted presentation of internalized antigens to CD8(+) T lymphocytes, a process called cross-presentation. In dendritic cells (DC), the only antigen-presenting cells that activate naive T cells, cross-presentation is particularly efficient after internalization of opsonized antigens or immune complexes, which are cross-presented through a proteasome- and transporter associated with antigen processing (TAP)-dependent MHC class I antigen presentation pathway. We now show that FcgammaR-mediated cross-presentation is tightly regulated during DC maturation. Cross-presentation increases soon after activation by lipopolysaccharides, and it is then inhibited in fully mature cells. The initial induction of cross-presentation results from an increase of both antigen internalization and delivery to the cytosol, and from a slight rise in the activity of the proteasome and TAP. The subsequent block of cross-presentation in mature DC is a consequence of the selective down-modulation of antigen internalization and cytosolic delivery, while proteasome and TAP activities continue to rise. Therefore, FcgammaR-mediated cross-presentation is regulated during DC maturation by the selective control of antigen internalization and transport to the cytosol.