A novel single-nucleotide polymorphism (SNP), 829C-->T in the 3'-untranslated region of the human dihydrofolate reductase (DHFR) gene transcript, was identified in the study population of 37 patients with childhood leukemias/lymphomas and 83 healthy Japanese children. Frequencies of the DHFR 829C/C, 829C/T, and 829T/T genotypes were 83.8, 10.8, and 5.4%, respectively, in the cases and 74.7, 19.3, and 6.0% in the controls, showing no significant difference in genotype frequencies between the cases and controls. When determined by real-time quantitative reverse transcription-PCR analysis, the highest expression of the DHFR transcript was demonstrated in the samples with a DHFR 829T/T polymorphism (P < 0.001). Direct association of the presence of the SNP with methotrexate-related adverse events in each patient was not demonstrated in this limited analysis. These data suggest that the novel DHFR 829 polymorphism is associated with a positive role in gene expression and provide evidence of a functional SNP in the 3' regulatory region of the gene.