© 2014, Sharma et al.; licensee Springer. Background: Epithelial ovarian cancer (EOC) is characterized by the overexpression of cancer antigen 125 (CA125), a mucinous glycoprotein that serves as a tumor biomarker. Early diagnosis of EOC is plagued by its asymptomatic nature of progression and the limitations of currently used immunoassay techniques that detect CA125 as a shed antigen in serum samples. Presently, there is no technique available for the in vivo evaluation of CA125 expression in malignant tissues. Moreover, there could be an unexplored pathophysiological time window for the detection of CA125 in EOC, during which it is expressed on tumor cells prior to being shed into the bloodstream. A method for the in vivo evaluation of CA125 expression on ovarian neoplasms earlier along disease progression and/or recurrence can potentially contribute to better disease management. To this end, the present work utilizes an anti-CA125 monoclonal antibody (MAb) and a single-chain variable fragment (scFv) labeled with the positron-emitting radionuclide 64Cu for preclinical molecular imaging of CA125 expression in vivo.Methods: Anti-CA125 MAb and scFv were prepared and functionally characterized for target binding prior to being tested as radiotracers in a preclinical setting.Results: Immunoblotting, immunofluorescence, and flow cytometry revealed specific binding of CA125-targeting vectors to NIH:OVCAR-3 cells and no binding to antigen-negative SKOV3 cells. 64Cu-labeled anti-CA125 MAb and scFv were obtained in specific activities of 296 and 122 MBq/mg, respectively. Both radioimmunoconjugate vectors demonstrated highly selective binding to NIH:OVCAR-3 cells and virtually no binding to SKOV3 cells. In vivo radiopharmacological evaluation using xenograft mouse models injected with 64Cu-labeled anti-CA125 MAb provided a standardized uptake value (SUV) of 5.76 (29.70 %ID/g) in OVCAR3 tumors 24 h post-injection (p.i.) versus 1.80 (5.91 %ID/g) in SKOV3 tumors. 64Cu-labeled anti-CA125 scFv provided an SUV of 0.64 (3.21 %ID/g) in OVCAR3 tumors 24 h p.i. versus 0.25 (1.49 %ID/g) in SKOV3 tumors. Results from small-animal PET imaging were confirmed by ex vivo autoradiography and immunohistochemistry.Conclusions: Radiolabeling of anti-CA125 MAb and scFv with 64Cu did not compromise their immunoreactivity. Both radioimmunoconjugates presented specific tumor uptake and expected biological clearance profiles. This renders them as potential immuno-PET probes for targeted in vivo molecular imaging of CA125 in EOC.