We evaluated the role of aberrant crypt foci (ACF) as biomarkers of colon cancer by studying the sequential development (6-28 weeks) from early lesion to tumor in the colon of azoxymethane-exposed F344 rats (15 mg/kg bw x 2). Surface examination of unsectioned methylene blue-stained colon preparations, transilluminated in the inverse light microscope, revealed two types of early lesions: classic elevated ACF and small flat lesions, which we denoted flat ACF and which were characterized by bright blue staining, compressed crypt openings, and crypts not elevated above the surrounding mucosa. At a later stage, the crypts surrounding large flat ACF became enlarged, a change that slightly raised the structure; principally, large flat ACF and nascent tumors displayed the same surface morphology. Furthermore, flat ACF with 18.6 +/- 10.6 crypt/focus and tumors showed a uniform picture of severe dysplasia with frequent presence of Paneth cells, compressed crypts, cytoplasmic/nuclear overexpression of beta-catenin, and nuclear overexpression of cyclin D1. In contrast, classic elevated ACF with 5.3 +/- 2.5 crypts/focus did not display such changes: they showed mainly hyperplasia, mild or moderate dysplasia but never severe dysplasia. Along the time course, the number of flat ACF + tumors, including microscopic and macroscopic, was virtually constant, approximately 2.5 lesions/rat. The number of classic elevated ACF was initially approximately 180 lesions/rat and terminally approximately 80 lesions/rat. Flat ACF grew significantly faster than classic elevated ACF. In conclusion, our data indicate a continuous developmental growth from small flat dysplastic ACF to the stage of a tumor. In contrast, classic elevated ACF do not seem to be as closely related to tumorigenesis.