The purpose of this study was to demonstrate the efficacy of an enrichment protocol for the detection of circulating carcinoma cells in the bloodstream of patients with various urologic cancers. Using 16-ml peripheral blood samples (BS) the mononuclear cells were isolated by density gradient centrifugation. The CD45 leukocyte depletion method based on a previous study was slightly modified and semi-automated by an immunomagnetic cell separation unit (autoMACS). Enriched tumor cells were analyzed on a single slide by cytokeratin (CK) immunocytochemistry. The number of recovered DU-145 prostate cancer cells in various spiking experiments was 70-88%. By the optimized tumor cell enrichment protocol 186 BS originated from 128 patients with different urologic cancers (60 prostate carcinoma, 34 bladder cancers, 24 renal cell carcinoma and 10 other tumors) were investigated before, during and after tumor surgery. In 59 BS from 52 patients on average 5 tumor cells were detected in each BS containing tumor cells. The median number of identified tumor cells was 8 cells per BS and patient. Tumor cells were found for the 3 tumor types with representative BS numbers in 29-39% of the investigated BS and in 38-53% of the affected patients. The detection rates increased in the order prostate carcinoma < renal cell carcinoma < bladder cancer. Surprisingly, in four bladder tumor cases with identified disseminated tumor cells in BS, the histopathological examination of the transurethral resection of bladder tumor specimens showed no evidence for tumor cells in situ but the affected patients had clinically known and histologically defined tumor residue or a bladder tumor recurrence during the follow-up. The semi-automated CD45 autoMACS depletion protocol for the enrichment and the detection of disseminated tumor cells in the peripheral bloodstream allows to study up to 20 BS per working day prospectively by one technician. The improved sensitivity and specificity might be of importance when applying the protocol to BS in future clinical studies.