Macrophages can promote tumor development. Preclinically, targeting macrophages by colony-stimulating factor 1 (CSF1)/CSF1 receptor (CSF1R) monoclonal antibodies (mAbs) enhances conventional therapeutics in combination treatments. The physiological distribution and tumor uptake of CSF1R mAbs are unknown. Therefore, we radiolabeled a murine CSF1R mAb and preclinically visualized its biodistribution by PET. CSF1R mAb was conjugated to N-succinyl-desferrioxamine (N-suc-DFO) and subsequently radiolabeled with zirconium-89 (⁸⁹Zr). Optimal protein antibody dose was first determined in non-tumor-bearing mice to assess physiological distribution. Next, biodistribution of optimal protein dose and ⁸⁹Zr-labeled isotype control was compared with PET and ex vivo biodistribution after 24 and 72 h in mammary tumor-bearing mice. Tissue autoradiography and immunohistochemistry determined radioactivity distribution and tissue macrophage presence, respectively. [⁸⁹Zr]Zr-DFO-N-suc-CSF1R-mAb optimal protein dose was 10 mg/kg, with blood pool levels of 10 ± 2% injected dose per gram tissue (ID/g) and spleen and liver uptake of 17 ± 4 and 11 ± 4%ID/g at 72 h. In contrast, 0.4 mg/kg of [⁸⁹Zr]Zr-DFO-N-suc-CSF1R mAb was eliminated from circulation within 24 h; spleen and liver uptake was 126 ± 44% and 34 ± 7%ID/g, respectively. Tumor-bearing mice showed higher uptake of [⁸⁹Zr]Zr-DFO-N-suc-CSF1R-mAb in the liver, lymphoid tissues, duodenum, and ileum, but not in the tumor than did ⁸⁹Zr-labeled control at 72 h. Immunohistochemistry and autoradiography showed that ⁸⁹Zr was localized to macrophages within lymphoid tissues. Following [⁸⁹Zr]Zr-DFO-N-suc-CSF1R-mAb administration, tumor macrophages were almost absent, whereas isotype-group tumors contained over 500 cells/mm². We hypothesize that intratumoral macrophage depletion by [⁸⁹Zr]Zr-DFO-N-suc-CSF1R-mAb precluded tumor uptake higher than ⁸⁹Zr-labeled control. Translation of molecular imaging of macrophage-targeting therapeutics to humans may support macrophage-directed therapeutic development.