Increased proliferative activity has been described frequently in the colons of animals treated with colon carcinogens and of patients at increased risk of colon cancer; it has been proposed as an intermediate biomarker of colon cancer. Aberrant crypt foci, microscopic lesions identified in whole-mount preparations of colons, are thought to be putative pre-neoplastic lesions. The present studies were carried out to evaluate the proliferative activity of aberrant crypt foci at several different time periods, and of tumors after a single dose of azoxymethane (AOM) in F344 rats. Rats were injected with 5-bromo-2'-deoxyuridine (BUdR) 1 hr before killing. Aberrant crypt foci and tumors were identified and marked in the whole-mount specimens, embedded in glycol methacrylate, and evaluated for histochemically demonstrable hexosaminidase activity. Hexosaminidase is known to be altered in over 95% of aberrant crypt foci. Serial sections were evaluated for BUdR incorporation immunohistochemically with a monoclonal antibody. The mean proliferative activity of aberrant crypt foci in the distal colons was found to be increased 3- to 4-fold over that of the adjacent normal crypts at every time period analyzed (4 to 36 weeks) and was comparable to that seen in benign and malignant colon tumors in the same animals. The observed increase in proliferative activity further supports the hypothesis that aberrant crypt foci are putative pre-neoplastic lesions. Similar aberrant crypt foci, identified in human colons at increased risk of colon cancer, may provide important biomarkers for this common human cancer.