The anti-human epidermal growth factor receptor 2 (HER2/neu) antibody trastuzumab is administered to patients with HER2/ neu-overexpressing breast cancer. Whole-body noninvasive HER2/neu scintigraphy could help to assess and quantify the HER2/neu expression of all lesions, including nonaccessible metastases. The aims of this study were to develop clinical-grade radiolabeled trastuzumab for clinical HER2/neu immunoPET scintigraphy, to improve diagnostic imaging, to guide antibodybased therapy, and to support early antibody development. The PET radiopharmaceutical 89 Zr-trastuzumab was compared with the SPECT tracer 111 In-trastuzumab, which we have tested in the clinic already. Methods: Trastuzumab was labeled with 89 Zr and (for comparison) with 111 In. The minimal dose of trastuzumab required for optimal small-animal PET imaging and biodistribution was determined with human HER2/neu-positive or -negative tumor xenograft-bearing mice. Results: Trastuzumab was efficiently radiolabeled with 89 Zr at a high radiochemical purity and specific activity. The antigen-binding capacity was preserved, and the radiopharmaceutical proved to be stable for up to 7 d in solvent and human serum. Of the tested protein doses, the minimal dose of trastuzumab (100 mg) proved to be optimal for imaging. The comparative biodistribution study showed a higher level of 89 Zr-trastuzumab in HER2/neu-positive tumors than in HER2/neu-negative tumors, especially at day 6 (33.4 6 7.6 [mean 6 SEM] vs. 7.1 6 0.7 percentage injected dose per gram of tissue). There were good correlations between the small-animal PET images and the biodistribution data and between 89 In-trastuzumab uptake in tumors (R 2 5 0.972). Conclusion: Clinical-grade 89 Zr-trastuzumab showed high and HER2/neu-specific tumor uptake at a good resolution.