Mutation within the adult human stem cell (SC) compartment has been proposed as a factor in the initiation and promotion of carcinogenesis. Isolation of these cancer stem cells (CSCs) has proven difficult, limiting their subsequent phenotypic, functional, and genetic characterization. We have used the Hoechst 33342 dye efflux technique to isolate an epithelial side population (SP) from genitourinary (GU) cancers, which is enriched for cells with SC traits. With informed consent, samples were taken from patients with primary tumors and undergoing surgery for prostatic (CaP), invasive bladder transitional cell (TCC), and renal cell carcinomas (RCC). Single cell epithelial suspensions were extracted from these and incubated with Hoechst 33342. Hoechst SP/non-SP profiles were then generated by flow cytometry using standardized protocols. SP/non-SP cell cycle status was established by Hoechst 33342 and Pyronin Y staining. Immunocytochemistry staining was performed for markers suggested as stem markers as well as lineage-specific markers. Functionality was determined using colony-forming assays and long-term monolayer culture. A characteristic verapamil-sensitive SP was isolated from all 3 urological malignancies and represented 0.57% +/- 0.11% (CaP), 0.52% +/- 0.49% (TCC), and 5.9% +/- 0.9% (RCC) of the total epithelial population. Cell cycle analysis showed that the SP had enhanced numbers of cells in G(0) as compared to the total cell population (CaP 12.4% +/- 3.2 vs. 3.8% +/- 1.0, RCC 23.2% +/- 3.4 vs. 1.8% +/- 0.9, and TCC 28.5% +/- 4.9 vs. 4% +/- 1.3). Immunocytochemistry demonstrated an increased expression of proliferative and putative stem markers within the SP fraction. Cultures confirmed significant enhancement of colony-forming ability and proliferative capacity of the SP fraction. A characteristic SP enriched for stem-like cells has been isolated from the 3 most common urological malignancies. This provides strong evidence that Hoechst 33342 efflux is a conserved and unified mechanism in GU cancer.