Purpose CD103, an integrin specifically expressed on the surface of cancer-reactive T cells, is significantly increased during successful immunotherapy across human malignancies. In this study, we describe the generation and zirconium-89 (89 Zr) radiolabeling of monoclonal antibody (mAb) clones that specifically recognize human CD103 for non-invasive immune positron-emission tomography (PET) imaging of T cell infiltration as potential biomarker for effective anticancer immune responses. Experimental design First, to determine the feasibility of anti-CD103 immuno-PET to visualize CD103-positive cells at physiologically and clinically relevant target densities, we developed an 89 Zr-anti-murine CD103 PET tracer. Healthy, non-tumor bearing C57BL/6 mice underwent serial PET imaging after intravenous injection, followed by ex vivo biodistribution. Tracer specificity and macroscopic tissue distribution were studied using autoradiography combined with CD103 immunohistochemistry. Next, we generated and screened six unique mAbs that specifically target human CD103 positive cells. Optimal candidates were selected for 89 Zr-anti-human CD103 PET development. Nude mice (BALB/cOlaHsd-Foxn1nu) with established CD103 expressing Chinese hamster ovary (CHO) or CHO wild-type xenografts were injected with 89 Zr-anti-human CD103 mAbs and underwent serial PET imaging, followed by ex vivo biodistribution. Results 89 Zr-anti-murine CD103 PET imaging identified CD103-positive tissues at clinically relevant target densities. For human anti-human CD103 PET development two clones were selected based on strong binding to the CD103 + CD8 + T cell subpopulation in ovarian cancer tumor digests, non-overlapping binding epitopes and differential CD103 blocking properties. In vivo, both 89 Zr-anti-human CD103 tracers showed high target-to-background ratios, high target site selectivity and a high sensitivity in human CD103 positive xenografts. Conclusion CD103 immuno-PET tracers visualize CD103 T cells at relevant densities and are suitable for future non-invasive assessment of cancer reactive T cell infiltration.