Prostate cancer cells are heterogeneous in their tumorigenicity. For example, the side population cells isolated from LAPC9 xenografts are 100 to 1,000 times more tumorigenic than the corresponding non-side population cells. Highly purified CD44(+) prostate cancer cells from several xenografts are also enriched in prostate cancer stem/progenitor cells. Because the CD44(+) prostate cancer cell population is still heterogeneous, we wonder whether we could further enrich for tumorigenic prostate cancer cells in this population using other markers. Integrin alpha2beta1 has been proposed to mark a population of normal human prostate stem cells. Therefore, we first asked whether the alpha2beta1(+/hi) cells in prostate tumors might also represent prostate cancer stem cells. Highly purified (> or =98%) alpha2beta1(+/hi) cells from three human xenograft tumors, Du145, LAPC4, and LAPC9, show higher clonal and clonogenic potential than the alpha2beta1(-/lo) cells in vitro. However, when injected into the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse prostate or s.c., the alpha2beta1(+/hi) prostate cancer cells are no more tumorigenic than the alpha2beta1(-/lo) cells. Immunofluorescence studies reveal that CD44 and alpha2beta1 identify an overlapping and inclusive population of prostate cancer cells in that approximately 70% of alpha2beta1(+/hi) cells are CD44(+) and 20% to 30% of CD44(+) cells are distributed in the alpha2beta1(-/lo) cell population. Subsequently, we sorted out CD44(+)alpha2beta1(+/hi), CD44(+)alpha2beta1(-/lo), CD44(-)alpha2beta1(+/hi), and CD44(-)alpha2beta1(-/lo) cells from LAPC9 tumors and carried out tumorigenicity experiments. The results revealed a hierarchy in tumorigenic potential in the order of CD44(+)alpha2beta1(+/hi) approximately CD44(+)alpha2beta1(-/lo) > CD44(-)alpha2beta1(+/hi) >> CD44(-)alpha2beta1(-/lo). These observations together suggest that prostate cancer cells are organized as a hierarchy.