Methylenetetrahydrofolate reductase (MTHFR) is a key regulatory enzyme in the metabolism of folate, a nutrient which has recently been found to be inversely related to breast cancer in women who drink alcohol. Two common variants in the MTHFR gene (C677T and A1298C) have been associated with a reduced activity of this enzyme, thereby increasing the availability of folate for thymidylate and purine synthesis. We investigated the relationship of these variants with invasive breast cancer in a case-control study of 1,189 cases and 2,414 controls nested within the Multiethnic Cohort Study. The Multiethnic Cohort Study is a large prospective study of men and predominantly postmenopausal women of Japanese, White, African American, Latino, and Native Hawaiian origin, residing in Hawaii and Los Angeles. We found an overall nonsignificant, weak inverse association between breast cancer risk and the 677TT genotype and no association with the 1298C variant. The odds ratio [OR and 95% confidence interval (95% CI)] for the 677CC, 677CT, and 677TT genotypes were 1.00, 0.98 (0.83-1.15), and 0.86 (0.67-1.09), respectively. Those for the 1298AA, 1298AC, and 1298CC genotypes were 1.00, 0.93 (0.79-1.08), and 1.20 (0.88-1.65), respectively. However, the inverse association with the 677TT genotype was stronger (OR, 0.62; 95% CI 0.39-0.98) among women who were on hormone replacement therapy (HRT) at baseline, and the increased breast cancer risk due to HRT was not observed in women with the 677TT genotype. An increased breast cancer risk was suggested for alcohol intake >10 g/d, when compared with nondrinkers, but only among HRT users with the 677CC genotype (OR, 1.51; 95% CI, 0.96-2.37). Folate intake exhibited no modifying effect on the genotype-breast cancer relationship. These findings suggest that the MTHFR 677TT genotype may confer a 40% decreased breast cancer risk in postmenopausal women using HRT. This is consistent with the role of MTHFR in facilitating the flow of folate for thymidylate and purine synthesis and with the increased nucleic acid need resulting from the hyperproliferative effect of HRT on mammary epithelial cells.