We have developed a piglet model to assess chemotherapy administration directly into the fourth ventricle as a potential treatment for medulloblastoma and other malignant posterior fossa tumors. The objective of this study was to assess safety and pharmacokinetics after methotrexate infusions into the fourth ventricle. Catheters were inserted into the fourth ventricle and lumbar cistern in five piglets. Two milligrams of Methotrexate (MTX) was infused into the fourth ventricle on five consecutive days. Safety was assessed by neurological examination, 4.7 T MRI, and post-mortem pathological analysis. MTX levels in serum and cerebrospinal fluid (CSF) were measured, and area under the concentration-time curve (AUC) was calculated for CSF samples. No neurological deficits were caused by MTX infusions. One piglet died from complications of anesthesia induction for MRI scanning. MRI scans showed accurate catheter placement without signal changes in the brainstem or cerebellum. One piglet had asymptomatic ventriculomegaly. Pathological analysis demonstrated meningitis and choroid plexitis consisting predominantly of CD-3 positive T-lymphocytes in all piglets and a small focal area of subependymal necrosis in one. In all piglets, mean peak MTX level in fourth ventricular CSF exceeded that in lumbar CSF by greater than five-fold. Serum MTX levels were undetectable or negligible. Statistically significant differences between fourth ventricle and lumbar AUC were detected at peaks (P = 0.01) and at all collection time points (P = 0.01) but not at troughs (P = 0.36). MTX can be infused into the fourth ventricle without clinical or radiographic evidence of damage. An inflammatory response without clinical correlate is observed. Significantly higher peak MTX levels are observed in the fourth ventricle than in the lumbar cistern.