Twenty-nine children with acute lymphocytic leukemia were given 24-hr infusions of intermediate-dose methotrexate (MTX, 1000 mg/m2) with and without intrathecal (IT) MTX (12 mg/m2), followed by leucovorin rescue. There was substantial interpatient variability in MTX systemic clearance (98.3 ± 51 ml/min/m2), inducing total steady-state serum MTX concentrations ranging from 5.4 to 33.7 μM. The cerebrospinal fluid (CSF) concentration at the end of the infusion was 0.27 (±0.1) μM when no IT-MTX was given and correlated with total steady-state (24-hr) serum concentration of MTX. By stepwise regression, the CSF MTX concentration correlated better with the nonprotein bound (free) steady-state serum MTX concentration (r = 0.66, P < 0.01) than with total steady-state serum MTX concentration. Mean CSF: serum MTX concentration ratio was 0.023 (±0.04) when no IT MTX was given. When an IT MIX dose (12 mg/m2) was given at the start of the MIX infusion, the steady-state CSF MTX concentration was 1.1 (±0.4) μM, leading to a mean CSF: serum ratio of 0.073 (±0.05). Despite 7-hydroxy-MTX serum concentrations exceeding MIX concentrations immediately after infusion, 7-hydroxy-MTX was not detectable in CSF of most patients (21 of 29), and was <50% of the concurrent MTX concentration when detectable. These data establish the substantial interpatient variability in CSF distribution of MTX after intermediate-dose MTX infusions and establish a significant correlation between steady-state free concentration of MTX in serum and CSF MTX concentration.