Methylation of the retinoic acid receptor-beta2 (RARbeta2) P2 promoter is hypothesized to be an important mechanism for loss of RARbeta2 function during early mammary carcinogenesis. The frequency of RARbeta2 P2 methylation was tested in (a) 16 early stage breast cancers and (b) 67 random periareolar fine needle aspiration (RPFNA) samples obtained from 38 asymptomatic women who were at increased risk for breast cancer. Risk was defined as either (a) 5-year Gail risk calculation > or = 1.7%; (b) prior biopsy exhibiting atypical hyperplasia, lobular carcinoma in situ, or ductal carcinoma in situ; or (c) known BRCA1/2 mutation carrier. RARbeta2 P2 promoter methylation was assessed at two regions, M3 (-51 to 162 bp) and M4 (104-251 bp). In early stage cancers, M4 methylation was observed in 11 of 16 (69%) cases; in RPFNA samples, methylation was present at M3 and M4 in 28 of 56 (50%) and 19 of 56 (38%) cases, respectively. RPFNAs were stratified for cytologic atypia using the Masood cytology index. The distribution of RARbeta2 P2 promoter methylation was reported as a function of increased cytologic abnormality. Methylation at both M3 and M4 was observed in (a) 0 of 10 (0%) of RPFNAs with Masood scores of < or = 10 (nonproliferative), (b) 3 of 20 (15%) with Masood scores of 11 to 12 (low-grade proliferative), (c) 3 of 10 (30%) with Masood scores of 13 (high-grade proliferative), and (d) 7 of 14 (50%) with Masood scores of 14 of 15 (atypia). Results from this study indicate that the RARbeta2 P2 promoter is frequently methylated (69%) in primary breast cancers and shows a positive association with increasing cytologic abnormality in RPFNA.