Lymphatic mapping for prostate cancer can be used to determine therapeutic strategies. Sentinel node visualization requires sufficient nodal tracer uptake. We evaluated the effect of an increase in particle concentration on preoperative and intraoperative sentinel node depiction. Enrolled in the study were 50 consecutive patients with prostate cancer. The first 25 patients (group A) received nanocolloid with standard labelling (0.4 ml (99m)Tc per 0.1 mg nanocolloid). The last 25 patients (group B) received nanocolloid with a reduced labelling dilution volume (0.4 ml (99m)Tc per 0.2 mg nanocolloid). The aimed injected volume and dosage were the same for both groups (225 MBq in 0.4 ml). Intratumoral tracer injection was followed by planar lymphoscintigraphy (15 min and 2 h), SPECT/CT and laparoscopic sentinel lymphadenectomy. Lymph node visualization was evaluated using a four-point scoring system (0 nonvisualization to 3 intense visualization) and count quantification on the 2-h anterior lymphoscintigram. In addition to the gamma ray detection probe, a portable gamma camera was used for intraoperative sentinel node visualization. Preoperative visualization in group A was 88% (mean 2.0 sentinel nodes per patient) versus 100% in group B (mean 2.6 sentinel nodes per patient). Visualization scores (p=0.008), total counts (p=0.001) and maximum counts per pixel (p=0.034) in the sentinel nodes were significantly better in group B. This also led to more efficient intraoperative detection of the sentinel nodes with the portable gamma camera (84% in group A versus 100% in group B). Enhancement of the particle concentration may lead to significant improvement in sentinel node visualization and intraoperative localization in patients with prostate cancer. Further research regarding optimization of radiotracer labelling by changing the particle concentration is warranted.