Alpha-klotho is a single-pass membrane protein primarily expressed by the kidneys. Klotho deficiency in chronic kidney disease contributes to an accelerated aging phenotype. We report here development of [89Zr]DFO-anti-klotho positron emission tomography (PET) imaging as a novel non-invasive method for assessing whole-body alpha-klotho distribution. Rat monoclonal anti-mouse klotho antibody was reacted with SCN-Bn-deferoxamine (DFO) and was radiolabeled using Zirconium-89. In vitro testing of [89Zr]DFO-anti-mKlotho was done in a distal convoluted tubule kidney cell line and with 40-micron whole kidney sections from C57BL/6J mice. Competitive binding was assessed in co-incubation studies with unlabeled anti-mKlotho antibody. For in vivo testing, C57BL/6J mice were injected retro-orbitally with [89Zr]DFO-anti-mKlotho and were scanned using Inveon PET/CT. Autoradiographs of kidney sections were obtained post-imaging on select animals. Radiochemical yield of [89Zr]DFO-anti-mKlotho was >70% and radiochemical purity was confirmed by iTLC. Specific binding in the kidney cell line was reduced by 60% in the presence of unlabeled anti-mKlotho. In the PET/CT scans, initial uptake of [89Zr]DFO-anti-mKlotho was observed in the intestines and liver. Selective retention of radioactivity was observed in the kidneys in the subsequent 24, 48, and 72 hrs scans with cortical binding of [89Zr]DFO-anti-mKlotho clearly visualized. Sites of lower alpha-klotho expression were not visualized. In summary, we have successfully synthesized [89Zr]DFO-anti-mKlotho and our initial in vitro and in vivo studies in mice demonstrate selective binding in the kidney cortex, which is known to express high levels of alpha-klotho. PET imaging promises to be a novel tool for in vivo evaluation of alpha-klotho distribution.