The methylenetetrahydrofolate reductase (MTHFR) 677C-->T polymorphism is thought to influence the partitioning of 1-carbon units between methylation and other components of 1-carbon metabolism and to influence the risk and etiology of several major cancers and cardiovascular disease. Our objective was to determine the effect of the MTHFR 677C-->T polymorphism and folate status on the relative rate and extent of in vivo synthesis of DNA precursors. Adequately nourished, healthy women (9 CC, 9 TT) were infused with [3-(13)C]serine and [(13)C(5)]methionine for 9 h before and after 7 wk of consumption of a low-folate diet. Blood was drawn over 5 d for monocyte DNA isolation. Isotopic enrichment of the nucleosides in DNA digests was determined by LC-MS/MS. Maximum thymidine enrichment tended to be higher (P = 0.07) in TT than in CC subjects, suggestive of marginally higher mean thymidylate synthesis. However, the subset of TT subjects who exhibited formyltetrahydrofolate in erythrocytes (an indicator of 1-carbon partitioning) had greater (P = 0.036) thymidine enrichment than CC subjects, who had no erythrocyte formyltetrahydrofolate. Purine enrichment was not affected by genotype or folate depletion. However, the deoxyadenosine to deoxyguanosine enrichment ratio was significantly higher in TT subjects, suggesting a greater relative rate of adenine synthesis. The approximately 40% greater (P = 0.012) labeling of the methyl group of methyldeoxycytidine during folate depletion suggests a change in the origin of this 1-carbon unit. This is the first time that 1-carbon incorporation into human DNA has been measured in vivo after infusion of (13)C-labeled 1-carbon precursors. These findings support the feasibility of further assessment of factors affecting deoxynucleotide synthesis and DNA methylation in human 1-carbon metabolism.