Vitamin B-12 (cobalamin) is a cofactor for only two enzymes, methionine synthase and L-methylmalonyl-CoA mutase. The serum vitamin B-12 concentration has been shown to have limitations in specificity and sensitivity in diagnosing vitamin B-12 deficiency and predicting response to therapy in subjects with clinical deficiency syndromes. Serum methylmalonic acid and/or total homocysteine concentrations have been shown to be elevated in almost every patient who has a clinical response to vitamin B-12. In elderly populations serum methylmalonic acid concentrations are elevated in the majority (60-66%) of subjects who have elevated total homocysteine concentrations, suggesting that vitamin B-12 deficiency (with or without associated folate deficiency) and/or chronic renal insufficiency may be the primary cause of most of the elevated total homocysteine concentrations in elderly populations. In such subjects vitamin B-12 and folate concentrations are both frequently in the low or low normal range, making differentiation of the clinical syndromes by use of serum vitamin concentrations problematic. Elevations of 2-methylcitric acid and cystathionine also result from vitamin B-12 deficiency. Serum N-methylglycine concentrations are normal in cobalamin deficiency but are increased in 40% of patients deficient in folate. In conclusion, elevations of methylmalonic acid and total homocysteine are very sensitive and specific in diagnosing vitamin B-12 deficiency and can be used to help differentiate vitamin B-12 deficiency from folate deficiency. Elevated total homocysteine concentrations that may have been attributed to folate deficiency in elderly subjects may in many instances be the result of vitamin B-12 deficiency even though serum vitamin B-12 concentrations are within normal limits.