The staging lymph node dissection in patients with penile carcinoma is accompanied with a high morbidity. As many patients are free of nodal metastases the lymphoscintigraphic sentinel node biopsy is supposed to minimize perioperative morbidity in these patients. In the current study the accuracy of the lymphoscintigraphic sentinel node biopsy was verified against the gold standard of radical inguinal dissection. In particular, patients with enlarged lymph nodes have also been included since one half of these patients is known to have histologically negative lymph nodes. Between 2000 and 2004 fifteen patients with penile carcinoma were elected to undergo bilateral groin dissection, thus 30 inguinal areas have been dissected. Nine patients have had clinically palpable nodes. All patients underwent lymphoscintigraphy after injection of Tc99-nanocolloid subcutaneously into the peritumoral area. Intraoperatively the sentinel nodes were identified with the aid of a gamma ray detection probe and excised. Afterwards a standard groin dissection was performed and the different lymph nodes were histopathologically assessed separately. In all patients lymph nodes with high radioactivity uptake were detected bilaterally. In 10 out of 30 inguinal areas histopathologically positive lymph nodes were present. In four of them the sentinel node was positive for tumor but in six dissection areas lymph node metastases were found despite a negative sentinel node. These patients had clinically palpable lymph nodes in their histologically positive inguinal regions. If no palpable nodes were present dynamic sentinel biopsy detected the positive nodes. The current study showed that dynamic sentinel node biopsy in patients with clinically suspicious lymph nodes is of low value for detection of lymphatic spread in penile cancer. Therefore the gold standard in these patients remains the radical groin dissection. However, dynamic sentinel node biospy is still a promising strategy to identify lymphatic spreading in clinically N0 patients and therefore to prevent unnecessary groin dissection.