Purpose: The value of measuring expression of individual genes relevant to particular chemotherapy drugs and encoding metabolizing enzymes, transporters, or drug targets, as predictors of treatment response and outcome in pediatric acute lymphoblastic leukemia (ALL), remains controversial. Experimental Design: In a case-control population of 91 pediatric B-precursor ALL patients [42 relapsed within 4 years (cases) and 49 did not relapse (controls)], we used real-time reverse transcription- PCR to measure transcript levels for 20 genes relevant to chemotherapy with the five major drugs used to treat this disease, including asparaginase, 6-mercaptopurine, methotrexate, prednisone, and vincristine. Results were confirmed in a separate case-control population of 26 patients. Results: Only the human reduced folate carrier (hRFC) gene, encoding the major membrane transporter for methotrexate, showed a significant difference in median transcript levels between the 42 cases and the 49 controls (P = 0.0278, Wilcoxon test). Using cutoffs for hRFC expression levels (based on Akaike information criterion), there were statistically significant associations between hRFC transcripts and treatment relapse (P = 0.0052). hRFC-B, corresponding to the major hRFC transcript form in ALL, was also measured by real-time reverse transcription-PCR and was prognostic. The association between treatment relapse and hRFC levels was validated in a separate study population of 14 cases and 12 controls from an earlier case-control study (P = 0.0221). Conclusions: Our results strongly suggest the prognostic importance of hRFC gene expression to treatment outcomes in pediatric ALL. They validate our previous studies of hRFC transcriptional regulation in pediatric ALL and provide further compelling evidence for the critical role for methotrexate in the successful treatment of this disease.