The C677T variant of methylenetetrahydrofolate reductase (MTHFR), a key enzyme in the remethylation of homocysteine to methionine, is a frequent genetic cause of mild hyperhomocysteinemia among individuals with low folate status. However, little is known about the influence of subject characteristics, such as age and sex, on the relation between the C677T MTHFR polymorphism and fasting plasma total homocysteine (tHcy) concentrations. The aim of the present study was to explore the influence of age and gender, together with folate status, on the association between the C677T polymorphism and tHcy concentrations. The C677T genotype was determined for 1820 participants from the fifth examination of the Framingham Offspring Study. Mean age of the participants was 56 y (range 28-82 y). The allelic distribution was not different from the Hardy-Weinberg equilibrium, with a TT frequency comparable in men and women (14%). Geometric mean tHcy was 15% higher in men than in women (P < 0.001), and women had significantly higher plasma folate levels (P < 0.001). Geometric mean tHcy was significantly higher in TT participants (P = 0.001) than in participants with the CC and CT genotypes among those with plasma folate <12.5 nmol/L, but not among those with higher folate status. Because of a significant age and sex interaction (P = 0.02), we further stratified the low folate group by age and sex, and observed that the association between genotype and tHcy was confined to men <55 y old (P < 0.001). Our results suggest that age and sex modify the contribution of the MTHFR C677T mutation to fasting tHcy concentrations.