The metastatic status of sentinel lymph nodes (SLNs) might be the most important prognostic factor in breast cancer. In this paper, we report to our knowledge the first study of 99m Tc-rituximab as a radiotracer for imaging of SLNs using lymphoscintigraphy in both preoperative and intraoperative breast cancer patients. Methods: 99m Tcrituximab was designed as an SLN tracer targeting the CD20 antigen, which expresses extensively in LNs. A retrospective study was performed on 2,317 patients with primary breast cancer who underwent lymphoscintigraphy and sentinel lymph node biopsy (SLNB). Before imaging, all patients were administered a preoperative peritumoral injection of 37 MBq of 99m Tc-rituximab. Results: 99m Tc-rituximab was synthesized in both high radiolabeling yield and high radiochemical purity (.95%), with molecular integrity and immune activity well maintained. The initial study of 100 breast cancer patients showed that the success rate of SLN lymphoscintigraphy by injection of 99m Tc-rituximab, as compared with SLNB, was 100%, and the sensitivity, specificity, accuracy, and false negative rate were 97.4%, 100%, 98.0%, and 2.60%, respectively. Of the following 2,217 patients studied, the success rate of lymphoscintigraphy and SLNB was 98.8% and 99.9%, and the average number of SLN was 1.78 (range, 1-10) and 2.85 (range, 1-15). Age was an independent predictor of the number of SLNs identified by lymphoscintigraphy and intraoperative handheld γ-probe (P , 0.05), and other factors-such as sex, imaging time, primary tumor site, histopathologic subtype, clinical T stage, and immunochemistry-were not (P . 0.05). However, the SLN metastatic rates were different in patients with different histopathologic subtype, clinical T stage, and immunochemistry (P , 0.05). Conclusion: Here we report the first study of the new radiotracer 99m Tc-rituximab for breast cancer lymphoscintigraphy. This tracer showed great feasibility, safety, and effectiveness for SLN mapping in breast cancer patients.