In penile cancer there is still a diagnostic dilemma between over treatment of lymph node-negative patients and the missing of occult metastases by watchful waiting. In the current study the value of fluorescence diagnosis during radical inguinal lymph node dissection was evaluated. Five patients with penile cancer were elected to undergo groin dissection. All patients received 5-aminolevulinic acid (5-ALA) orally before the operation for fluorescence diagnosis. Intraoperatively, fluorescence detection of the lymph nodes was performed by visual detection and spectroscopy. Two of the five patients had positive inguinal lymph nodes. Fluorescence in tumor-bearing tissue was detectable in the exposed lymph nodes. Protoporphyrin IX (PPIX) is accumulated in tumor-positive lymph nodes, making fluorescence diagnosis in penile cancer possible. More studies with higher patient numbers are necessary to evaluate optimal dosage and excitation conditions to detect tumor-bearing nodes in vivo.