Folate deficiency, either by diet or drug, increases plasma homocysteine(Hcy). Elevated concentrations of Hcy are injurious to cerebrovascular endothelium, and hyperhomocysteinemia is a risk factor for stroke. Excess Hcy can be metabolized to excitatory amino acid (EAA) neurotransmitters, such as homocysteic acid (HCA) and cysteine sulfinic acid (CSA), which are implicated in the pathogenesis of seizures and excitotoxic neuronal death. We postulated that excess Hcy and EAA neurotransmitters may be in part the mediators of methotrexate (MTX) associated neurotoxicity. We utilized high-performance liquid chromatography to measure the content of Hcy, HCA, CSA, and 8 other amino acids in the cerebrospinal fluid (CSF) of two groups (treatment; control) of individuals. We defined the treatment group to include patients who received MTX within one week of a scheduled lumbar puncture. Individuals without any MTX exposure and those who received MTX greater than one week before a scheduled lumbar puncture comprised the control groups. The patients in the treatment group had significantly (p=0.006) greater concentrations of Hcy in CSF (0.815μM ± 0.215 [SEM], n=23) than did patients in the control group (0.171μM ± 0.038, n=22). HCA and CSA were not detectable in the CSF of control patients (n=14); however, MTX produced marked accumulation of CSF HCA (118.9μM ± 32.2, n=16) and CSA (26.4μM± 7.8, n=16) in the treatment group. No changes in the CSF concentrations of the 8 other amino acids were detected, connoting a specific effect on Hcy and related compounds. Patients who had clinical signs of neurologic toxicity at the time of lumbar puncture had many of the highest concentrations of Hcy, HCA, and CSA. These data indicate that MTX increases CSF Hcy and EAAs, supporting our hypothesis that MTX neurotoxicity may be mediated by biochemical folate deficiency, leading to elaboration and accumulation of Hcy and excitotoxic neurotransmitters.