Aging is associated with increased incidence of colon cancers. It is also becoming evident that cancer stem cells (CSC) play a vital role in the pathogenesis and prognosis of colon cancer. Recently, we reported the presence of colon cancer stem-like cells in macroscopically normal mucosa in patients with adenomatous polyps and that they increase with aging, suggesting that aging may predispose the colon to carcinogenesis. In the current study we have examined the combined effects of aging and carcinogen exposure on the status of colon CSCs in an experimental model. We used Young (4-6 months) and aged (22-24 months) rats and exposed them to the carcinogen, dimethylhydroxide (DMH). We investigated the expression of colon cancer stem cell markers, CD44, CD166, EpCam. and ALDH1 as well as EGFR expression in normal colonic crypt epithelium following carcinogen treatment. Our results demonstrate that aging per se or Carcinogen treatment alone causes an increase in the number of colon cancer stems cells, as evidenced by increased immunoreactive-CSC-markers positive cells in the colonic mucosa. In aged rats, Carcinogen exposure results in a more pronounced increase in colon cancer stem cells. Our study shows that in aging colon the effects of carcinogens are more pronounced, and an increase in colon CSCs is one of the earliest changes preceding tumor development. Moreover, the Current investigation of the use of a panel of immunohistochemical markers of colon CSC can potentially serve as a prognostic marker during screening for colon cancer. Published by Elsevier Inc.