Purpose To determine whether shortening the infusion duration of high-dose methotrexate (HDMTX; 1 g/m²) affects the in vivo accumulation of active methotrexate polyglutamates (MTXPG_1-7) in leukemia cells and whether this differs among major acute lymphoblastic leukemia (ALL) subtypes. Methods From June 2000 through October 2007, 356 children with ALL were randomly assigned to receive initial single-agent treatment with HDMTX (1 g/m²) as either a 24-hour infusion or a 4-hour infusion at two pediatric hospitals in the United States. The primary outcome measures were the accumulation of MTXPG_1-7 in leukemia cells and the antileukemic effects (eg, inhibition of de novo purine synthesis in bone marrow ALL cells, and decrease in circulating ALL cells). Results The 24-hour infusion resulted in significantly higher amounts of MTXPG_1-7 in bone marrow leukemia cells (median: 1,695 v 1,150 pmol/10⁹ cells, P = .0059), and better antileukemic effects. The 24-hour infusion had the greatest effect on MTXPG_1-7 accumulation in hyperdiploid ALL (median: 3,919 v 2,417 pmol/10⁹ cells, P = .0038); T-cell ALL exhibited smaller differences in MTXPG_1-7 but greater antileukemic effects with the longer infusion (median decrease in leukemia cells: 88.4% v 51.8%, P = .0075). In contrast, infusion duration had no significant impact on MTXPG_1-7 accumulation or antileukemic effects in ALL with the t(12;21)/(ETV6-RUNX1) chromosomal translocation. Conclusion Shortening the infusion time of HDMTX reduces accumulation of active methotrexate in leukemia cells and decreases antileukemic effects, with differing consequences among major ALL subtypes.