The transforming growth factor-beta (TGF-beta) type II receptor (RII) is a colon cancer suppressor gene that is inactivated by mutation in 90% of human colon cancers arising via the microsatellite instability (MSI) pathway of carcinogenesis. To determine the pathophysiological consequence of RII mutations, we have determined the timing of their onset among 22 MSI human colon adenomas of varying stages. No RII mutations were detected in any early MSI adenoma, including all those with simple tubular or villous histology. The earliest RII mutation detected was in a region of high-grade dysplasia but was absent from the surrounding simple adenoma. Six additional RII mutations were all found in highly progressed adenomas that contained regions of frankly invasive adenocarcinoma. These RII mutations were detected in both the advanced adenomas and their adjacent regions of carcinoma. RII mutation is a late event in MSI adenomas and correlates tightly with progression of these adenomas to cancer.