Folates are carriers of one-carbon units and are metabolized by 5,10-methylenetetrahydrofolate reductase (MTHFR) and other enzymes that use riboflavin, cobalamin, or vitamin B6 as cofactors. These B vitamins are essential for the remethylation and transsulfuration of homocysteine, which is an important intermediate in one-carbon metabolism. We studied the MTHFR 677C-->T polymorphism and B vitamins as modulators of one-carbon metabolism in 10,601 adults from the Norwegian Colorectal Cancer Prevention (NORCCAP) cohort, using plasma total homocysteine (tHcy) as the main outcome measure. Mean concentrations of plasma tHcy were 10.4 micromol/liter, 10.9 micromol/liter, and 13.3 micromol/liter in subjects with the CC (51%), CT (41%), and TT (8%) genotypes, respectively. The MTHFR 677C-->T polymorphism, folate, riboflavin, cobalamin, and vitamin B6 were independent predictors of tHcy in multivariate models (P<.001), and genotype effects were strongest when B vitamins were low (P<or=.006). Conversely, the MTHFR polymorphism influenced B vitamin effects, which were strongest in the TT group, in which the estimated tHcy difference between subjects with vitamin concentrations in the lowest compared with the highest quartile was 5.4 micromol/liter for folate, 4.1 micromol/liter for riboflavin, 3.2 micromol/liter for cobalamin, and 2.1 micromol/liter for vitamin B6. Furthermore, interactions between B vitamins were observed, and B vitamins were more strongly related to plasma tHcy when concentrations of other B vitamins were low. The study provides comprehensive data on the MTHFR-B vitamin network, which has major effects on the transfer of one-carbon units. Individuals with the TT genotype were particularly sensitive to the status of several B vitamins and might be candidates for personalized nutritional recommendations.