The purpose of this study was to determine the safety, distribution, internal dosimetry, and initial human epidermal growth factor receptor 2 (HER2)–positive tumor images of ⁶⁴Cu-DOTA-trastuzumab in humans. Methods: PET was performed on 6 patients with primary or metastatic HER2-positive breast cancer at 1, 24, and 48 h after injection of approximately 130 MBq of the probe ⁶⁴Cu-DOTA-trastuzumab. Radioactivity data were collected from the blood, urine, and normal-tissue samples of these 6 patients, and the multiorgan biodistribution and internal dosimetry of the probe were evaluated. Safety data were collected for all the patients after the administration of ⁶⁴Cu-DOTA-trastuzumab and during the 1-wk follow-up period. Results: According to our results, the best timing for the assessment of ⁶⁴Cu-DOTA-trastuzumab uptake by the tumor was 48 h after injection. Radiation exposure during ⁶⁴Cu-DOTA-trastuzumab PET was equivalent to that during conventional ¹⁸F-FDG PET. The radioactivity in the blood was high, but uptake of ⁶⁴Cu-DOTA-trastuzumab in normal tissues was low. In 2 patients, ⁶⁴Cu-DOTA-trastuzumab PET showed brain metastases, indicative of blood–brain barrier disruptions. In 3 patients, ⁶⁴Cu-DOTA-trastuzumab PET imaging also revealed primary breast tumors at the lesion sites initially identified by CT. Conclusion: The findings of this study indicated that ⁶⁴Cu-DOTA-trastuzumab PET is feasible for the identification of HER2-positive lesions in patients with primary and metastatic breast cancer. The dosimetry and pharmacologic safety results were acceptable at the dose required for adequate PET imaging.