© 2016, World Molecular Imaging Society. Purpose: The purpose of the present study is to evaluate safety, human radiation dosimetry, and optimal imaging time of [89Zr]trastuzumab in patients with HER2-positive breast cancer. Procedures: Twelve women with HER2-positive breast cancer underwent [89Zr]trastuzumab positron emission tomography (PET)/X-ray computed tomography (CT) twice within 7 days post-injection. Biodistribution data from whole-torso PET/CT images and organ time-activity curves were created using data from all patients. Human dosimetry was calculated using OLINDA with the adult female model. Results: High-quality images and the greatest tumor-to-nontumor contrast were achieved with images performed 5 ± 1 day post-injection. Increased [89Zr]trastuzumab uptake was seen in at least one known lesion in ten patients. The liver was the dose-limiting organ (retention of ∼12 % of the injected dose and average dose of 1.54 mSv/MBq). The effective dose was 0.47 mSv/MBq. No adverse effects of [89Zr]trastuzumab were encountered. Conclusion: [89Zr]trastuzumab was safe and optimally imaged at least 4 days post-injection. The liver was the dose-limiting organ.