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We performed a feasibility study to determine the optimal dosage and time of administration of the monoclonal antibody zirconium-89 (89Zr)-trastuzumab to enable positron emission tomography (PET) imaging of human epidermal growth factor receptor 2 (HER2)-positive lesions. Fourteen patients with HER2-positive metastatic breast cancer received 37 MBq of 89Zr-trastuzumab at one of three doses (10 or 50 mg for those who were trastuzumab-naive and 10 mg for those who were already on trastuzumab treatment). The patients underwent at least two PET scans between days 2 and 5. The results of the study showed that the best time for assessment of 89Zr-trastuzumab uptake by tumors was 4-5 days after the injection. For optimal PET-scan results, trastuzumab-naive patients required a 50 mg dose of 89Zr-trastuzumab, and patients already on trastuzumab treatment required a 10 mg dose. The accumulation of 89Zr-trastuzumab in lesions allowed PET imaging of most of the known lesions and some that had been undetected earlier. The relative uptake values (RUVs) (mean ± SEM) were 12.8 ± 5.8, 4.1 ± 1.6, and 3.5 ± 4.2 in liver, bone, and brain lesions, respectively, and 5.9 ± 2.4, 2.8 ± 0.7, 4.0 ± 0.7, and 0.20 ± 0.1 in normal liver, spleen, kidneys, and brain tissue, respectively. PET scanning after administration of 89Zr-trastuzumab at appropriate doses allows visualization and quantification of uptake in HER2-positive lesions in patients with metastatic breast cancer.