Hypermethylation of tumor suppressor and other regulatory genes is thought to play an important role in colorectal neoplasia and tumorigenesis. This study examined the association between gene methylation status in baseline adenomas and subsequent adenoma recurrence in a randomized dietary intervention study, the Polyp Prevention Trial. The methylation status of four genes [CDKN2A (p16), PTGS2 (COX2), ESR1 (ER-alpha), and PGR(PR)] was determined by MethyLight in 284 baseline adenomas from 196 trial participants. The association of gene methylation with recurrence was determined using logistic regression models. Gene methylation was evaluated as percent of methylated reference, a measure of methylation of each gene relative to control DNA. ESR1methylation status was inversely associated with adenoma recurrence, odds ratio = 0.36 (95% confidence interval, 0.15-0.88; P = 0.02) for the highest compared with the lowest quartile of the ESR1methylation. Further, ESR1 methylation status was inversely associated with the recurrence of multiple adenomas, advanced adenomas, and the recurrence of adenomas in the proximal but not distal bowel. No association between CDKN2A, PTGS2, or PGR methylation and adenoma recurrence was observed. These data suggest that ESR1 methylation may play a role in subsequent adenoma recurrence.