In multiple pregnancies, demands for folic acid are considerably increased. The most common inborn error of folate metabolism is mild methylenetetrahydrofolate reductase (MTHFR) deficiency due to the synthesis of a thermolabile variant of the enzyme with impaired catalytic activity which leads to reduced 5-methyltetrahydrofolate (5-methyl-THF) and mildly elevated homocysteine plasma concentrations when folate status is inadequate. To investigate whether the number of offspring is influenced by this mutation, we determined the frequency of the 677C-->T substitution in 156 singleton and 40 twin mothers with dichorionic placentation. The T allele frequency in singleton (0.30) and twin mothers (0.16) was significantly different (P = 0.011). Mothers with the 677C-->T mutation had a 2.28 times lower risk of having a twin pregnancy than those without (95% confidence interval = 1.18-4.66; P = 0.008). Our observation would explain, at least in part, the hereditary trait of multiple gestations and is in agreement with the ethnic distribution pattern of the T allele which has been found to be inversely correlated with the incidence of dichorionic twins. Our findings suggest that the MTHFR 677C-->T mutation interferes with human brood size, probably by influencing the proliferation of rapidly dividing embryonic and maternal cells.