Cytotoxic immunoglobulin G antibodies are an essential component of therapeutic approaches aimed at depleting self-reactive or malignant cells. More recent evidence suggests that the tissue in which the target cell resides influences the underlying molecular and cellular pathways responsible for cytotoxic antibody activity. By studying cytotoxic IgG activity directed against natural killer cells in primary and secondary immunological organs, we show that distinct organ-specific effector pathways are responsible for target cell depletion. While in the bone marrow, the classical complement pathway and the high-affinity Fcγ-receptor I expressed on organ-resident macrophages were both involved in removing opsonized target cells; in the spleen and blood, all activating FcγRs but not the classical complement pathway were critical for target cell killing. Our study suggests that future strategies aimed at optimizing overall cytotoxic antibody activity may need to consider organ-specific pathways to achieve a maximal therapeutic effect. Display omitted •Distinct organ-specific pathways are responsible for cytotoxic IgG activity•Antibody dose dictates FcγR versus complement involvement in IgG activity•FcγRI and the classical complement pathway mediate IgG activity in the bone marrow•Organ-resident macrophages mediate IgG activity in spleen and bone marrow Cytotoxic antibodies are essential therapeutic tools for depleting autoreactive or malignant cells. Gordan et al. show that the organ environment in which target cell killing takes place and the antibody dose dictate which molecular and cellular pathways contribute to cytotoxic IgG activity in vivo.