Choline is involved in the synthesis of phospholipids, including blood lipids, and is the immediate precursor of betaine, which serves as a methyl group donor in a reaction converting homocysteine to methionine. Several cardiovascular risk factors are associated with plasma homocysteine, whereas little is known about their relationship to choline and betaine. We examined the relation of plasma choline and betaine to smoking, physical activity, BMI, percent body fat, waist circumference, blood pressure, serum lipids, and glucose in a population-based study of 7074 men and women aged 47-49 and 71-74 y. Overall plasma concentrations (means +/- SD) were 9.9 +/- 2.3 micromol/L for choline and 39.5 +/- 12.5 micromol/L for betaine. Choline and betaine were lower in women than in men and in younger subjects compared with older (P < 0.0001). Multivariate analyses showed that choline was positively associated with serum triglycerides, glucose, BMI, percent body fat, waist circumference (P < 0.0001 for all), and physical activity (P < 0.05) and inversely related to HDL cholesterol (P < 0.05) and smoking (P < 0.0001). Betaine was inversely associated with serum non-HDL cholesterol, triglycerides, BMI, percent body fat, waist circumference, systolic and diastolic blood pressure (P < 0.0001 for all), and smoking (P < 0.05) and positively associated with HDL cholesterol (P < 0.01) and physical activity (P < 0.0001). Thus, an unfavorable cardiovascular risk factor profile was associated with high choline and low betaine concentrations. Choline and betaine were associated in opposite directions with key components of metabolic syndrome, suggesting a disruption of mitochondrial choline dehydrogenase pathway.