OBJECTIVE: To investigate the usefulness of presurgical image fusion of three-dimensional single photon emission computed tomography (SPECT) with computed tomography/magnetic resonance imaging (CT/MRI) for detecting primary prostatic lymphatic landing sites (sentinel lymph nodes [SLNs]) using an intraoperative gamma probe in patients undergoing radical prostatectomy for organ-confined prostate cancer after intraprostatic (99m)Tc-nanocolloid injection. METHODS: In 15 patients the surgeon was unaware of the preoperative SPECT/CT/MRI fusion imaging results. In 21 patients the surgeon was supported by the preoperative SPECT/CT/MRI images. Selective SLN resection was followed by an extended backup pelvic lymph node dissection (PLND) and radical prostatectomy. RESULTS: In the group of 15 patients (gamma probe-guided SLN search only) presurgical SPECT/CT/MRI identified 137 SLNs (median of 10/patient; range: 2-19) and a total of 135 SLNs (median of 8/patient; range: 2-20) were detected intraoperatively (detection rate 98.5%). In the group of 21 patients with the surgeon knowing the preoperative SPECT/CT/MRI findings, 189 (median of 9/patient; range: 3-20) of the 190 imaged SLNs (median of 8/patient, range: 2-19) were found intraoperatively (detection rate 99.5%). Median operating time for sentinel lymphadenectomy using gamma probe only was 100 min (range: 40-130) versus 70 min (range: 20-140) for the SPECT/CT/MRI-supported resection (p=0.025). A median of 9 SLNs per patient (range: 2-19) was confirmed histologically. CONCLUSIONS: Preoperative SPECT/CT/MRI is highly reliable to detect nonmetastatic sentinel nodes of the prostate. The operating time for SPECT/CT/MRI-supported SLN resection is significantly reduced compared to SLN resection guided by gamma probe alone. Both procedures take significantly longer than routine extended PLND.