Folate, vitamin B6, vitamin B12, methionine, choline, and betaine are nutrients involved in one-carbon metabolism and have been hypothesized to reduce the risk of breast cancer. However, previous epidemiologic studies on most of these nutrients and breast cancer risk have been inconclusive and have included primarily postmenopausal women. No study has examined choline and betaine in relation to breast cancer risk. Therefore, we examined the intake of these nutrients in relation to breast cancer risk among 90,663 premenopausal women ages 26 to 46 years in 1991 in the Nurses' Health Study II. Nutrient intake was assessed with a validated food frequency questionnaire in 1991, 1995, and 1999. During 12 years of follow-up from 1991 to 2003, we documented 1,032 incident cases of invasive breast cancer. Overall, none of the nutrients was associated with risk of breast cancer. The results were similar by levels of alcohol intake and folate intake and for estrogen receptor-negative breast cancer. In conclusion, we found no evidence that higher intakes of nutrients involved in one-carbon metabolism reduce risk of breast cancer among premenopausal women.