Choline, an essential nutrient, is critical in maintaining biomembrane integrity (via phosphatidylcholine) and supplying methyl groups for one-carbon metabolism (via betaine). Choline adequate intakes were established for the first time in 1998. However, little is known about the impact of pregnancy and genetic variation on choline metabolism and requirements. The overall goal of my research was to quantify the effects of pregnancy, genetic variation, and choline intake on biomarkers of choline metabolism. To achieve this goal, two separate feeding studies that employed stable isotope methodology were conducted. Study 1 examined the effect of the methylenetetrahydrofolate reductase (MTHFR) 677C[RIGHTWARDS ARROW]T genetic variant, and choline intake, on biomarkers of choline metabolism. Study 2 investigated the effect of pregnancy, and choline intake, on biomarkers of choline metabolism. Deuterium labeled methyl-d9-choline was administered in both studies as the tracer. Study 1 demonstrated that the MTHFR 677TT (versus the 677CC) genotype favors the use of choline as a methyl donor. MTHFR 677TT genotype enhanced the conversion of choline to betaine. In addition, when a higher choline intake was consumed, more of the choline was converted to betaine as opposed to entering the CDP-choline pathway for phosphatidylcholine synthesis among men with MTHFR 677TT (versus 677CC) genotype. Study 2 demonstrated that pregnancy alters choline metabolism with 10 -60% lower circulating concentrations of choline derived methyl donors among pr egnant versus nonpregnant women. Stable isotope data suggested that pregnancy increased choline partitioning to the CDPcholine pathway at the expense of betaine synthesis, and also increased the use of cholinederived methyl groups for methionine synthesis and phosphatidylcholine synthesis through the phosphatidylethanolamine N-methyltransferase (PEMT) pathway. Despite the upregulation of both pathways for phosphatidylcholine synthesis, PEMT -phosphatidylcholine was selectively transferred to the fetus. Consumption of 930 (versus 480) mg choline/d increased circulating concentrations of choline derived methyl donors, restored the partitioning of choline between the CDP-choline and choline oxidative pathways to the nonpregnant state, and enhanced the use of choline as a methyl donor in both maternal and fetal compartments. In conclusion, choline requirements are elevated in those with the MTHFR 677TT genotype and among third trimester pregnant women, and current recommendations may be suboptimal for these population sub-groups.