Sentinel lymph node biopsy (SLNB) is used to detect axillary lymph node metastases in breast cancer. Preoperative radiocolloid injection with lymphoscintigraphy (PL) is performed before SLNB. Few comparisons between 1- and 2-day PL protocols exist. Opponents of a 2-day protocol have expressed concerns of radiotracer washout to nonsentinel nodes. Proponents cite lack of scheduling conflicts between PL and surgery. A total of 387 consecutive patients with clinically node-negative breast cancer underwent SLNB with PL. Lymphoscintigraphy images were obtained within 30 minutes of radiocolloid injection. Axillary lymph node dissection was performed if the sentinel lymph node (SLN) could not be identified. Data were collected regarding PL technique and results. In all, 212 patients were included in the 2-day PL group and 175 patients in the 1-day PL group. Lymphoscintigraphy identified an axillary sentinel node in 143/212 (67.5%) of patients in the 2-day group and 127/175 (72.5%) in the 1-day group (P = 0.28). SLN was identified at surgery in 209/212 (98.6%) patients in the 2-day group and 174/175 (99.4%) in the 1-day group (P = 0.41). An average of 3 SLN was found at surgery in the 2-day group compared with 3.15 in the 1-day group (P = 0.43). SLN was positive for metastatic disease in 54/212 (25.5%) patients in the 2-day group compared with 40/175 (22.9%) in the 1-day group (P = 0.55). A 2-day lymphoscintigraphy protocol allows reliable detection of the SLN, of positive SLN and equivalent SLN harvest compared with a 1-day protocol. The timing of radiocolloid injection before SLNB can be left at the discretion of the surgeon.