Introduction. Multiple radioactive lymph nodes are often removed during the course of sentinel lymph node (SLN) biopsy for breast cancer when both blue dye and radioactive colloid injection are used. Some of the less radioactive lymph nodes are second echelon nodes, not true SLNs. The purpose of this analysis was to determine whether harvesting these less radioactive nodes, in addition to the 'hottest' SLNs, reduces the false-negative rate. Methods. Patients were enrolled in this multicenter (121 surgeons) prospective, institutional review board-approved study after informed consent was obtained. Patients with clinical stage T1-2, N0, M0 invasive breast cancer were eligible. This analysis includes all patients who underwent axillary SLN biopsy with the use of an injection of both isosulfan blue dye and radioactive colloid. The protocol specified that all blue nodes and all nodes with 10% or more of the ex vivo count of the hottest node should be removed and designated SLNs. All patients underwent completion level I/II axillary dissection. Results. SLNs were identified in 672 of 758 patients (89%). Of the patients with SLNs identified, 403 patients (60%) had more than 1 SLN removed (mean, 1.96 SLN/patient) and 207 patients (31%.) had nodal metastases. The use of filtered or unfiltered technetium sulfur colloid had no impact on the number of SLNs identified. Overall, 33% of histologically positive SLNs had no evidence of blue dye staining. Of those patients with multiple SLNs removed, histologically positive SLNs were found in 130 patients. In 15 of these 130 patients (11.5 %), the hottest SLN was negative when a less radioactive node was positive for tumor. If only the hottest node had been removed, the false-negative rate would have been 13.0% versus 5.8% when all nodes with 10% or more of the ex vivo count of the hottest node were removed (P = .01). Conclusions. These data support the policy that all blue nodes and all nodes with 10% or more of the ex vivo count of the hottest SLN should be harvested for optimal nodal staging.