Aberrant crypt foci (ACF) in the colon have long been thought to be precancerous lesions and therefore monoclonal, but this is unresolved. Eleven ACF were isolated from five female patients. From these ACF, 178 individual aberrant crypts (ACs) were obtained and assessed for clonality using a method based on X chromosome inactivation of the polymorphic X-linked human androgen receptor (HUMARA) gene. Ten ACF were found to be mixtures of monoclonal and polyclonal types. The HUMARA analysis indicated that almost all ACF were polyclonal lesions. Simultaneously, we investigated K-ras mutations in each AC. We found that seven of the ACF harbored the K-ras mutation; strikingly, this was concordant for all of the ACs from a single ACF. These results, by contrast to the results of HUMARA analysis indicate that ACF lesions are monoclonal. This discrepancy suggests that ACF are apparently polyclonal because of de novo methylation on the active X chromosome. To confirm this possibility, we investigated the methylation status of the X chromosome in male ACF using a competitive PCR assay. One hundred nineteen individual ACs were isolated from eight ACF derived from four male patients. A total of 47 of 119 (39%) of male ACs showed de novo methylation of the HUMARA gene. We found that six of the eight male ACF harbored the K-ras mutation, and this was concordant for all of the ACs from a single ACF. We conclude that X chromosome methylation is unstable in ACF and that this might be an early event in colon carcinogenesis.