Introduction Bifunctional chelators have been shown to impact the biodistribution of monoclonal antibody (mAb)-based imaging agents. Recently, radiolabeled 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA)-peptide complexes have demonstrated improved in vivo stability and performance compared to their 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) counterparts. Here, we investigated if similar utility could be achieved with mAbs and compared 64 Cu-labeled DOTA and NODAGA-immunoconjugates for the detection of epithelial cell adhesion molecule (EpCAM) in a prostate cancer model. Methods DOTA and NODAGA-immunoconjugates of an EpCAM targeting mAb (mAb7) were synthesized and radiolabeled with 64 Cu (DOTA: 40°C for 1hr; NODAGA: 25°C for 1hr). The average number of chelators per mAb was quantified by isotopic dilution, and the biological activity of the immunoconjugates was evaluated by flow cytometry and ELISA. Radioligand assays were performed to compare cellular uptake and determine the dissociation constant ( K d ) and maximum number of binding sites ( B max ) for the immunoconjugates using DsRed-transfected PC3-cells. A PC3-DsRed xenograft tumor model was established in nude mice and used to perform biodistribution studies to compare organ uptake and pharmacokinetics. Results 64 Cu-DOTA-mAb7 and 64 Cu-NODAGA-mAb7 were prepared with chelator/protein ratios of 2–3 and obtained in comparable radiochemical yields ranging from 59 to 71%. Similar immunoreactivity was observed with both agents, and mock labeling studies indicated that incubation at room temperature or 40°C did not affect potency. 64 Cu-NODAGA-mAb7 demonstrated higher in vitro cellular uptake while 64 Cu-DOTA-mAb7 had higher K d and B max values. From the biodistribution data, we found similar tumor uptake (13.44±1.21%ID/g and 13.24±4.86%ID/g for 64 Cu-DOTA-mAb7 and 64 Cu-NODAGA-mAb7, respectively) for both agents at 24hr, although normal prostate tissue was significantly lower for 64 Cu-NODAGA-mAb7. 64 Cu-NODAGA-mAb7 also had less accumulation in the liver, suggesting excellent retention of the chelation complex in vivo . This was further confirmed by the higher blood activity of 64 Cu-NODAGA-mAb7, which corresponds to increased bioavailability afforded by the enhanced in vivo stability of the agent. Although tumor/muscle ratios were comparable, tumor/prostate ratios were >2-fold and 1.5-fold higher for 64 Cu-NODAGA-mAb7 at 24 and 48hr, respectively, and suggest better ability to discriminate tumor tissue with 64 Cu-NODAGA-mAb7 in our prostate cancer model. Conclusions To the best of our knowledge, this study represents the first comparison of 64 Cu-labeled DOTA and NODAGA immunoconjugates in vivo . Our results show favorable in vivo performance for 64 Cu-NODAGA-mAb7 which builds upon previous data on our hybrid mAb7 imaging agent by increasing the detection sensitivity for metastatic prostate tumors, as well as for other types of cancer that express EpCAM.