At present there are neither clinical nor experimental data available on the influence of technical details on the quality and reproducibility of prostate lymphoscintigraphy. Six adult fox hounds received repeated transrectal ultrasound guided intraprostatic injections of a technetium 99m labeled nanocolloid to prove the influence of different techniques of injection (one central injection in both prostate lobes vs two peripheral injections in both lobes) on tracer accumulation in sentinel lymph nodes (SLN) and other organs. The reproducibility of the favored technique was examined and in a last step it was subject to scrutiny following a reduction of the injected volume to 1% of the prostate volume. The number of scintigraphically visualized SLN varied between four and seven. They were located in the region of the internal and external iliac vessels, presacrally, paravesically, and directly paraprostatically. In five of six cases, the localization was reproducible both with the central application of an identical volume as well as with the volume reduced central injection. Tracer accumulation of SLNs and other organs varied enormously. We expect that with the combination of both injection techniques, even with the reduced injection volume, an optimized prostate lymphoscintigraphy will be the outcome.