Glucosephosphate isomerase (GPI), also known as phosphohexoisomerase, is a glycolytic enzyme whose activity is elevated in serum and CSF of patients with primary and metastatic CNS tumors. To improve the diagnostic accuracy of leptomeningeal metastasis (LM), we measured GPI levels in CSF of 66 patients with CNS or systemic malignancies with suspected LM. We determined GPI kinetically using a coupled enzyme reaction assay. There were 31 males and 35 females, aged 1 to seventy-six. Thirty-one had primary brain tumors, and 35 had systemic cancer with suspected CNS metastasis. We analyzed 95 samples; GPI values ranged from 0.85 to 329.0 U/l (normal, less than 20 U/l). Compared with positive CSF cytology and myelography, GPI sensitivity was 53.5% and specificity 92.1% for the group as a whole. There was a highly significant association between elevated CSF GPI (greater than 20 U/l) and LM. The results were similar for both primary CNS and systemic malignancies. Although not very sensitive, an elevated CSF GPI strongly suggests LM and may aid in early diagnosis of this serious complication of cancer.