Human and mammalian cells contain two cobalamin-dependent enzymes. Methylmalonyl-CoA mutase isomerizes L-methylmalonyl-CoA to succinyl-CoA in the propionyl-CoA pathway while methionine synthase catalyzes the transfer of the methyl group of 5-methyltetrahydrofolate to homocysteine to form methionine. Decreased activity of mutase leads to an increased methylmalonic acid in the serum of humans while decreased activity of methionine synthase leads to increased homocysteine in the serum of humans. In current studies of cultured fibroblasts, methylmalonic acid levels were specifically increased in media of normal fibroblasts exposed to propionate or in fibroblasts with mutations involving mutase. Homocysteine levels were specifically increased in media of normal fibroblasts exposed to reduced folate concentrations or in fibroblasts involving mutations of methionine synthase. In addition, exposure of normal cells to inhibitory cobalamin analogues resulted in an increase of both methylmalonic acid and homocysteine in the media. This method of analysis appears to be both specific and sensitive for reduced activity of these two enzymes in tissue culture.