Methylenetetrahydrofolate reductase (MTHFR) deficiency is an autosomal recessive disorder typically characterized by less than 20% of normal enzymatic activity, hyperhomocysteinemia, hypomethioninemia, and a range of neurological and vascular abnormalities. Nine rare mutations in severe MTHFR deficiency have been reported. In addition, a common missense mutation, which is associated with a milder deficiency and a thermolabile enzyme, has been observed in 35% of chromosomes from the general population, and may represent a risk factor for cardiovascular disease and for neural tube defects. This polymorphism may also be present in patients with mutations for severe MTHFR deficiency. Here we report a new MTHFR mutation identified by SSCP analysis and direct sequencing. The mutation is a G to A substitution at bp 1172 which converts a glycine to an aspartic acid residue. Since this mutation eliminates a recognition site for HaeIII, restriction digestion was used to detect this mutation in 4 of 15 patients with severe MTHFR deficiency. Thus it is the most frequent severe mutation found to date. Each of the 4 patients had one copy of the mutation while 42 controls were negative. Notably, 3 of the 4 patients had relatively late onset disease presenting at ages 13, 15, and 40 years, respectively. The fourth presented with seizures at age 7 months. Of the 4, only this infant was homozygous for the polymorphism associated with a thermolabile enzyme. The mother of this early-onset patient had had a stroke at age 4 years, but no other obvious findings of MTHFR deficiency. She presumably has one mutation for severe MTHFR deficiency and at least one copy of the thermolabile polymorphism. Studies are in progress to identify other mutations in these patients and to examine possible interactions of the polymorphism with heterozygosity or homozygosity for severe MTHFR mutations.