Background: Prostate cancer is the most common male malignancy in western society. It has been suggested that a small population of cells (tumour initiating cells) in prostate cancer harbour intrinsic characteristics that make them resistant to androgen deprivation treatment and chemotherapy. In this study, a small population of cells from the prostate cancer PC3 cell line were isolated as having high aldehyde dehydrogenase activity using ALDEFLUOR assay to assess whether they display the characteristics of tumour initiating cells. Methods: Real-time time PCR was performed on ALDHhigh, medium, low and ungated PC3 cells to detect the gene expression level of 10 potential tumor initiating cell markers. Sorted cells were also treated with the chemotherapy drugs vincristine, docetaxel and doxorubicin for 24 hours to assess drug resistance. In vivo, tumorigenicity studies were performed by injecting NOD/SCID mice with 5×104 ALDHhigh, low or ungated PC3 cells in the left flank. Tumour incidence and growth rate were recorded. Tumour morphology and angiogenesis were investigated using H&E and CD31 staining. Results: The embryonic stem cell marker oct-4 and the regulator of tumour suppressor genes DCLK1 were overexpressed in ALDHhigh cells which were also resistant to chemotherapy drugs. Our in vivo work showed that ALDHhigh cells had the ability to initiate tumours earlier with a shorter survival time. Tumours derived from ALDHhigh cells had increased angiogenesis and lower necrosis rates. Gene up-regulation of Trop2 and DCLK1 which are indicators of tumorigenicity was also observed in tumours arising from ALDHhigh cells.