We investigated the roles of age, vitamin B(12) markers, and the 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism as determinants of folate forms in serum. We measured the serum concentrations of (6S)-5-CH(3)-H(4)folate, (6S)-H(4)folate, (6S)-5-HCO-H(4)folate, (6R)-5,10-CH(+)-H(4)folate, and folic acid in 146 non-supplemented older participants (median age 74 years). The concentrations of total vitamin B(12), holotranscobalamin (holoTC), methylmalonic acid (MMA), and total homocysteine (tHcy) were also measured. Elevated metabolites (MMA > 271 nmol/L and tHcy > 12.0 μmol/L) were found in 24.0 and 63.0 % of the participants, respectively. We found a significant age-dependent decrease (participants with a median age of 87 years compared with participants with a median age of 60 years) in the sum of serum folate levels, the (6S)-5-CH(3)-H(4)folate concentration, and the (6S)-5-CH(3)-H(4)folate proportion. In addition, participants with elevated metabolite levels were older, had lower concentrations of the sum of folates and (6S)-5-CH(3)-H(4)folate, and had higher concentrations of (6S)-5-CHO-H(4)folate and creatinine but had a comparable holoTC/total vitamin B(12) ratio. No association was found between the MTHFR C677T genotype and serum folate forms. Low serum (6S)-5-CH(3)-H(4)folate concentrations and the proportion of (6S)-5-CH(3)-H(4)folate (percentage of the sum of folate forms) are related to older age and elevated MMA and tHcy levels.