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Folate is involved in DNA synthesis, repair, and methylation. It has been hypothesized that high intake of folate may reduce the risk of human cancers, including cancer of the breast. Using data from a population-based case-control study of breast cancer conducted in urban Shanghai during 1996-1998, we evaluated the association of dietary folate intake and breast cancer risk among 1321 cases and 1382 controls, 25-64 years of age, who never drank alcohol regularly or used vitamin supplements. Usual dietary habits were assessed with an in-person, interviewer-administered food frequency questionnaire developed and tested for use in this population. Unconditional logistic regression models were used to calculate odds ratios (ORs) and their 95% confidence intervals (95% CIs) after adjusting for potential confounding factors. Dietary folate intake was inversely associated with breast cancer risk (P for trend, 0.05) with an adjusted OR of 0.71 (95% CI, 0.56-0.92) observed among women who were in the highest quintile of intake. The inverse association was stronger after further adjusting for total fruit and vegetable and animal food intakes (OR, 0.62; 95% CI, 0.46-0.82; P for trend, 0.01). A more pronounced inverse association between folate intake and breast cancer risk (OR, 0.47; 95% CI, 0.25-0.88; P for trend, 0.01) was observed among women who consumed high levels of folate cofactors (methionine, vitamin B(12), and vitamin B(6)) than those whose intake levels of these nutrients were low. Dietary intake of methionine, vitamin B(12), and vitamin B(6) were not independently related to risk of breast cancer after adjusting for confounding factors. Thus, our study adds additional support to the protective role of dietary folate in breast carcinogenesis and suggests further that the effect of folate may be modified by dietary intake of methionine, vitamin B(12), and vitamin B(6).