In this study the nodal staging sensitivity of sentinel lymph node biopsy (SLNB) with detailed pathological and molecular biological examination has been investigated and compared to that of axillary lymph node dissection (ALND) with routine histological evaluation. Sentinel lymph nodes (SLNs) were removed by the dual-agent injection technique in 68 patients with primary, clinically node-negative breast cancer. Forty-seven patients had negative SLNs according to hematoxylin and eosin (H&E) staining. These H&E-negative SLNs were serially sectioned and examined at 250 microm levels by anticytokeratin immunohistochemistry (IHC). In 14 patients the SLNs were also investigated by cytokeratin 20 (CK20) reverse transcriptase polymerase chain reaction (RT-PCR). SLNB with IHC increased the node-positive rate by 26% (by 40% in tumors less than or equal to 2 cm in size (pT1) and by 9% in tumors more than 2 cm but less than or equal to 5 cm in size (pT2)). The sensitivity of SLNB with IHC was superior to that of ALND with routine histology in pT1 tumors and identical in pT2 tumors. The concordance between histology and RT-PCR was only 21%, and in two of three cases with positive histological results RT-PCR was negative. In conclusion, SLNB with detailed pathological and/or molecular biological evaluation can improve the sensitivity of regional staging. ALND can probably be abandoned in patients with pT1 SLN-negative breast cancer. Further prospective studies are required to determine the clinical significance of these detailed SLN evaluation techniques, but at present these methods are still investigational.