Purpose: Despite its clinical success, methotrexate (MTX) therapy is associated with toxicities such as seizures, the pathogenesis of which remains unclear. It has been suggested that hyperhomocysteinemia is caused by MTX and is responsible for its neurotoxic effects. The purposes of this study were to explore whether hyperhomocysteinemia was related to MTX administration and toxicity and whether homocysteine or MTX toxicity differed by methylenetetrahydrofolate reductase (MTHFR) or reduced folate carrier (RFC) genetic polymorphisms. Patients and Methods: We studied 53 children with newly diagnosed acute lymphoblastic leukemia who were consecutively treated on a single clinical protocol that included two courses of high-dose MTX (high-dose methotrexate [HDMTX]; 2.5 or 5.0 g/m² per day) as consolidation therapy. Results: The study participants’ median plasma homocysteine concentrations at 23 and 44 hours after HDMTX (9.00 μmol/L and 10.12 μmol/L, respectively) were greater than the concentrations immediately before HDMTX (5.77 μmol/L, P < .0001 for both comparisons). Seven days after HDMTX treatment, their plasma concentration returned to baseline. Nine patients experienced seizures, and five patients experienced thrombosis during the first 15 months of therapy, with a tendency for there to be higher plasma homocysteine in patients with seizures across all time points (P = .063) but not in patients with thrombosis (P = .59). We observed no significant differences in plasma or cerebrospinal fluid homocysteine levels or in toxicity based on the MTHFR 677C/T or RFC 80G/A genotypes. Conclusion: We conclude that homocysteine was transiently elevated after HDMTX and may be related to seizure risk in children with leukemia.