611 Background: While sentinel node biopsy (SNB) is well established as a minimally invasive means of staging the axilla in breast cancer patients, the optimal timing of injection of technetium 99m sulfur colloid (Tm) for SNB remains unclear. Methods: In a prospective multicenter study of 4131 patients who had a SNB for breast cancer followed by axillary node dissection, 3305 patients had a SNB using Tm with the elapsed time from injection to SNB being recorded. These 3305 patients formed the cohort of interest for this study. The dose of Tm remaining at the time of SNB was calculated with the formula mCiremaining=mCiinjected*0.5^(elapsed hours/6). Patients with injection of Tm ≤12h and >12h were compared using SPSS. Results: The mean age of the 3305 patients in this study was 60 years, with a mean tumor size of 1.8 cm. A sentinel node (SN) was identified in 95% of patients. SN identification (ID) was not affected by mCi injected (p=0.88), mCi remaining at time of SNB (p=0.13), or type of Tm (filtered vs. unfiltered, p=0.37). There was a statistically non-significant trend of more SN's removed in the ≤12h group (3.2 vs. 2.5, p=0.06). False negative rate was not affected by mCi injected (p=0.39), mCi remaining at time of SNB (p=0.24), or type of Tm (filtered vs. unfiltered, p=1.00). The overall false negative (FN) rate was 8.0%. Of the 3305 patients in this study, 3221 were injected ≤12h prior to SNB and 84 were injected >12h prior to SNB. For the patients injected ≤12h compared to >12h, there was no difference in SN ID rate, FN rate, counts of the hottest node, or background counts despite more mCi injected and less mCi remaining at SNB in the >12h cohort (see Table). Conclusions: Injecting Tm >12h prior to SNB has an acceptable SN identification rate, and the FN rate was not significantly different than injecting Tm ≤12h prior to SNB. [Table: see text] No significant financial relationships to disclose.