PURPOSE: Previous studies has demonstrated the utility of human epidermal growth factor receptor type 2 (HER2) as an attractive target for cancer molecular imaging and therapy. An affibody protein with strong binding affinity for HER2, Z, has been reported. Various methods of chelator conjugation for radiolabeling HER2 affibody molecules have been described in the literature including N-terminal conjugation, C-terminal conjugation, and other methods. Cu-64 has recently been extensively evaluated due to its half-life, decay properties, and availability. Our goal was to optimize the radiolabeling method of this affibody molecule with Cu-64, and translate a positron emission tomography (PET) probe with the best in vivo performance to clinical PET imaging of HER2-positive cancers. PROCEDURES: In our study, three anti-HER2 affibody proteins-based PET probes were prepared, and their in vivo performance was evaluated in mice bearing HER2-positive subcutaneous SKOV3 tumors. The affibody analogues, Ac-Cys-Z, Ac-Z(Cys), and Ac-Z-Cys, were synthesized using the solid phase peptide synthesis method. The purified small proteins were site-specifically conjugated with the maleimide-functionalized chelator, 1,4,7,10-tetraazacyclododecane-1,4,7-tris- aceticacid-10-maleimidethylacetamide (maleimido-mono-amide-DOTA). The resulting DOTA-affibody conjugates were then radiolabeled with Cu-64. Cell uptake assay of the resulting PET probes, [Cu]DOTA-Cys-Z, [Cu]DOTA-Z(Cys), and [Cu]DOTA-Z-Cys, was performed in HER2-positive human ovarian SKOV3 carcinoma cells at 4 and 37 °C. The binding affinities of the radiolabeled peptides were tested by cell saturation assay using SKOV3 cells. PET imaging, biodistribution, and metabolic stability studies were performed in mice bearing SKOV3 tumors. RESULTS: Cell uptake assays showed high and specific uptake by incubation of Cu-64-labeled affibodies with SKOV3 cells. The affinities (K) of the PET radio probes as tested by cell saturation analysis were in the low nanomolar range with the ranking of [Cu]DOTA-Cys-Z (25.2 ± 9.2 nM) ≈ [Cu]DOTA-Z-Cys (32.6 ± 14.7 nM) > [Cu]DOTA-Z(Cys) (77.6 ± 22.2 nM). In vitro stability and in vivo metabolite analysis study revealed that all three probes were stable enough for in vivo imaging applications, while [Cu]DOTA-Cys-Z showed the highest stability. In vivo small-animal PET further demonstrated fast tumor targeting, good tumor accumulation, and good tumor to normal tissue contrast of all three probes. For [Cu]DOTA-Cys-Z, [Cu]DOTA-Z(Cys), and [Cu]DOTA-Z-Cys, tumor uptake at 24 h are 4.0 ± 1.0 % ID/g, 4.0 ± 0.8 %ID/g, and 4.3 ± 0.7 %ID/g, respectively (mean ± SD, n = 4). Co-injection of the probes with non-labeled anti-HER2 affibody proteins confirmed in vivo specificities of the compounds by tumor uptake reduction. CONCLUSIONS: The three Cu-64-labeled Z analogues all display excellent HER2 targeting ability and tumor PET imaging quality. Although varied in the position of the radiometal labeling of these three Cu-64-labeled Z analogues, there is no significant difference in tumor and normal tissue uptakes among the three probes. [Cu]DOTA-Cys-Z stands out as the most superior PET probe because of its highest affinities and in vivo stability.