Prostate cancer cells include a small population of cancer stem-like/cancer initiating cells, which have roles in cancer initiation and progression. Recently aldehyde dehydrogenase activity was used to isolate stem cells of various cancer and normal cells. We evaluated the aldehyde dehydrogenase activity of the human prostate cancer cell line 22Rv1 (ATCC®) with the ALDEFLUOR® assay and determined its potency as prostate cancer stem-like/cancer initiating cells. The human prostate cancer cell line 22Rv1 was labeled with ALDEFLUOR reagent and analyzed by flow cytometry. ALDH1 high and ALDH1 low cells were isolated and tumorigenicity was evaluated by xenograft transplantation into NOD/SCID mice. Tumor sphere forming ability was evaluated by culturing in a floating condition. Invasion capability was evaluated by the Matrigel™ invasion assay. Gene expression profiling was assessed by microarrays and reverse transcriptase-polymerase chain reaction. ALDH1 high cells were detected in 6.8% of 22Rv1 cells, which showed significantly higher tumorigenicity than ALDH1 low cells in NOD/SCID mice (p <0.05). Gene expression profiling revealed higher expression of the stem cell related genes PROM1 and NKX3-1 in ALDH1 high cells than in ALDH1 low cells. ALDH1 high cells also showed higher invasive capability and sphere forming capability than ALDH1 low cells. Results indicate that cancer stem-like/cancer initiating cells are enriched in the ALDH1 high population of the prostate cancer cell line 22Rv1. This approach may provide a breakthrough to further clarify prostate cancer stem-like/cancer initiating cells. To our knowledge this is the first report of cancer stem-like/cancer initiating cells of 22Rv1 using the aldehyde dehydrogenase activity assay.