Children with Down syndrome (DS) and acute lymphoblastic leukemia (ALL) often suffer from severe toxicities during treatment, especially with high-dose methotrexate (HD-MTX). Systematic data on methotrexate (MTX) toxicity in these patients are rare. We analyzed seven MTX-associated toxicities during consolidation therapy in 103 DS- and 1,109 non-DS-patients (NDS) with ALL (NDS-ALL) enrolled in ALL-Berlin-Frankfurt-Münster (ALL-BFM) trials between 1995-2016 and 1995-2007, respectively. Patients received four courses MTX (5 g/m each) plus intrathecal MTX and 6-mercaptopurine (6-MP). From 2004 onwards, a dose of 0.5 g/m in the first MTX course has been recommended for DS-patients. DS-patients showed higher rates of grade 3/4 toxicities after the first course with 5 g/m MTX compared to NDS-patients (grade 3/4 toxicities 62 in 45 DS-patients 516 in 1,089 NDS-patients, <0.001). The dose reduction (0.5 g/m ) in DS-patients has reduced toxicity (39 in 51 patients, <0.001) without increasing the relapse risk (reduced dose, 5-year cumulative relapse incidence = 0.09±0.04 high dose, 0.10±0.05, =0.51). MTX dose escalation to 1.0 g/m for DS-patients who tolerated 0.5 g/m (n= 28 of 51 patients) did not result in an increased rate of grade 3/4 toxicities after the second course ( =0.285). Differences in MTX plasma levels at 42 and 48 hours after the start of the first methotrexate infusion did not explain higher toxicity rates in DS-patients treated with 0.5 g/m compared to NDS-patients treated with 5 g/m Within the DS cohort a higher MTX plasma level was associated with increased toxicity. In conclusion, dose reduction in the first MTX course reduced severe toxicities without increasing the risk of relapse. ( ).