The biology of the normal colonic mucosa suggests that colon cancer originates from normal colon stem cells. CD44 cancer stem cells have been identified in breast and prostate cancer, and we therefore examined whether CD44 similarly identified colon cancer stem cells. Initial assays found CD44hi colon tumor cells to have enhanced soft agar colony-forming ability. Subsequently, CD44hi cells isolated from 4 primary colon adenocarcinoma xenografts were found to be highly tumorigenic in immune deficient mice. CD44hi cells consistently formed tumors with 1,000 cells, and in multiple experiments, as few as 10 and 100 CD44hi cells formed tumors in 7/10 and 21/28 mice, respectively. In contrast, CD44− colon tumor cells were either nontumorigenic or 10–50-fold less tumorigenic. CD44hi cells could be serially passaged up to 4 times in vivo, suggesting self-renewal capacity, and formed tumors that recapitulated the heterogeneity of the original patient tumor. CD44hi cells were significantly enriched for nuclear activated β-catenin, a key element in normal stem/progenitor cells and in early colon tumor progression. Bromodeoxyuridine (BrdU) labeling studies indicated that CD44hi cells divide slowly relative to the CD44− cells, suggesting their tumorigenicity is not simply due to faster proliferation. Aldehyde dehydrogenase (ALDH) sort further increased the tumorigenicity of CD44hi cells from 2/2 patient tumors, but CD133 tumor cells in our hands did not have increased tumorigenicity. Our observations indicate that CD44 is a marker of stem-like cells in colon cancer, and support the use of additional markers to further purify colon cancer stem cells. © 2008 Wiley-Liss, Inc.