Women with human epidermal growth factor receptor 2 (HER2)–positive breast cancer are candidates for treatment with the anti-HER2 antibody trastuzumab. Assessment of HER2 status in recurrent disease is usually made by core needle biopsy of a single lesion, which may not represent the larger tumor mass or other sites of disease. Our long-range goal is to develop PET of radiolabeled trastuzumab for systemically assessing tumor HER2 expression and identifying appropriate use of anti-HER2 therapies. The purpose of this study was to evaluate PET/CT of ⁶⁴Cu-DOTA-trastuzumab for detecting and measuring tumor uptake of trastuzumab in patients with HER2-positive metastatic breast cancer. Eight women with biopsy-confirmed HER2-positive metastatic breast cancer and no anti-HER2 therapy for 4 mo or longer underwent complete staging, including ¹⁸F-FDG PET/CT. For 6 of the 8 patients, ⁶⁴Cu-DOTA-trastuzumab injection (364–512 MBq, 5 mg of trastuzumab) was preceded by trastuzumab infusion (45 mg). PET/CT (PET scan duration 1 h) was performed 21–25 (day 1) and 47–49 (day 2) h after ⁶⁴Cu-DOTA-trastuzumab injection. Scan fields of view were chosen on the basis of ¹⁸F-FDG PET/CT. Tumor detection sensitivity and uptake analyses were limited to lesions identifiable on CT; lesions visualized relative to adjacent tissue on PET were considered PET-positive. Radiolabel uptake in prominent lesions was measured as maximum single-voxel standardized uptake value (SUV_max). Liver uptake of ⁶⁴Cu was reduced approximately 75% with the 45-mg trastuzumab predose, without significant effect on tumor uptake. The study included 89 CT-positive lesions. Detection sensitivity was 77%, 89%, and 93% for day 1, day 2, and ¹⁸F-FDG, respectively. On average, tumor uptake was similar for ⁶⁴Cu-DOTA-trastuzumab and ¹⁸F-FDG (SUV_max and range, 8.1 and 3.0–22.5 for day 1 [n = 48]; 8.9 and 0.9–28.9 for day 2 [n = 38]; 9.7 and 3.3–25.4 for ¹⁸F-FDG [n = 56]), but same-lesion SUV_max was not correlated between the 2 radiotracers. No toxicities were observed, and estimated radiation dose from ⁶⁴Cu-DOTA-trastuzumab was similar to ¹⁸F-FDG. ⁶⁴Cu-DOTA-trastuzumab visualizes HER2-positive metastatic breast cancer with high sensitivity and is effective in surveying disseminated disease. A 45-mg trastuzumab predose provides a ⁶⁴Cu-DOTA-trastuzumab biodistribution favorable for tumor imaging. ⁶⁴Cu-DOTA-trastuzumab PET/CT warrants further evaluation for assessing tumor HER2 expression and individualizing treatments that include trastuzumab.