Polymorphism in 5,10-methylenetetrahydrofolate reductase (MTHFR), a central enzyme in folate metabolism, has been shown to affect the sensitivity of patients to folate-based drugs such as methotrexate. In this study, we investigated whether a common single nucleotide polymorphism at position 677 in the donor or recipient's MTHFR gene affects the risk for acute graft-versus-host disease (GVHD) following allogeneic hematopoietic stem cell transplantation (HSCT) from HLA-identical sibling donors when the recipient receives prophylactic treatment with methotrexate for GVHD. MTHFR genotypes were determined in 159 recipients with a hematological disease and their donors using polymerase chain reaction-restriction fragment length polymorphism analysis of genomic DNA. The 677TT genotype, which encodes an enzyme with approximately 30% of the activity of the wild-type (677CC), was observed in 13% of patients and in 8% of normal donors. Multivariate analyses demonstrated a significant association between 677TT genotype in patients and a lower incidence of grade I-IV acute GVHD (relative risk, 0.35; 95% confidence interval, 0.13-0.95; P = 0.040). There was no association between the incidence of acute GVHD and the donor MTHFR genotypes. These results suggest that greater immunosuppression by methotrexate due to low MTHFR enzyme activity decreases the risk of acute GVHD in recipients of allogeneic HSCT.