Purpose: For isotope mapping, many authors have defined the sentinel lymph node (SLN) as the one containing isotope counts higher than the axillary background. However, no study has sought to define an optimal guideline, and the number of SLNs needed for precise detection of metastatic SLNs is still a subject of debate. Accordingly, we set out to determine the optimal sampling number of SLNs. Methods: Between January 2005 and December 2008, we enrolled 1,026 patients with primary invasive breast cancer. These patients had received sentinel lymph node biopsy (SLNB) using radioisotopes at Yeungnam University Hospital. During SLNB, ex vivo isotope counts were measured for each SLN. And, the number and metastatic status of harvested SLNs were investigated. We defined SLNs as lymph nodes that show an isotope count โฅ10 times that of the axillary background. Each SLN was labeled as S1 (highest isotope count), S2 (second highest), or S3 and so on, in descending isotopecount-order. If an SLN was positive for metastasis, completion axillary lymph node dissection was performed. Results: The mean age of patients was 48.9 years (ranges, 22-83 years). The mean number of removed SLNs was 2.8 (ranges, 1-11). Of the 1,026 patients enrolled, 311 (30.3%) had SLN metastasis: S1 was positive (S1+) in 258 patients (82.9%), S2 was positive (S1-, S2+) in 40 patients (12.3%), and S3 was positive (S1-, S2-, S3+) in 13 patients (3.9%). There was no metastasis in SLNs defined as S4, S5 and so on. The average number of sampling SLNs until detecting metastasis in S1, S2, and S3 was 1.24 (1-4), 2.45 (2-4), and 3.46 (3-5). Conclusion: During SLNB done using radioisotopes, for accurate axillary lymph node staging, up to the 3rd isotope-count-order of SLNs should be removed. The average number of SLNs needed for detecting metastasis in S3 is four.