Purpose: Lymphoscintigraphy and sentinel node biopsy are used in detection of axillary lymph node metastasis in breast cancer patients, but standardized technique is not established. We compared the results of the injection the morning of surgery (1 day protocol) with the subareolar injection the day before surgery (2 day protocol) with the subareolar injection in patients with breast cancer having lymphoscintigraphy and sentinel node biopsy. Materials and Methods: This study included 349 patients who underwent the breast cancer operation during 2001-2004. One hundred seventy one patients (1 day protocol, 1 hour) was injected 0.8 ml of Tc-99m Tin-Colloid (37 MBq) by subareolar injection on the morning of surgery. One hundred seventy eight patients (2 day protocol, 16 hour) was injected 0.8 ml of Tc-99m Tin-Colloid (185 MBq) on the afternoon before surgery. Lymphoscintigraphy was performed in sitting position and sentinel node localization was performed by hand-held gamma probe during operation. Result: In the 1 day protocol, 153 cases (89.5%) of the sentinel node were localized by lymphoscintigraphy and 150 cases (87.7%) were localized by gamma probe. In the 2 day protocol, 159 cases (89.3%) were localized by lymphoscintigraphy and 154 cases (86.5%) were localized by gamma probe. There was no significant difference in localization of sentinel node between the 1 day and the 2 day protocol by lymphoscintigraphy and gamma probe (p>0.05, p>0.05). Conclusion: There was no difference the result of localization of sentinel node with subareolar injection between the 1 day and the 2 day protocol in breast cancer patients. Because the 2 day protocol allows the enough time of performing lymphoscintigraphy, it is more useful in localization of sentinel node in breast cancer patients. (Nucl Med Mol Imaging 2009;43(1):55-59)