The activity of the myelin-associated enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) was assayed in the cerebrospinal fluid (CSF) of 107 neurologic patients by a new and sensitive fluorometric method. The activity of CNP was about 20 nmol per hour per milligram protein or 12 nmol per hour per milliliter CSF. At these extremely low levels, the presence of even a small amount of blood (which has slightly greater activity) significantly elevated CNP values. Patients with radicular syndromes had slightly higher than average CNP activities, but there was no difference in enzyme activities of 47 patients with multiple sclerosis and the general neurologic population. CNP activity was not related to stage of demyelinating illness or intrathecal injection of steroid. CNP-like myelin basic protein may be released into the CSF after destruction of myelin, but our results suggest that the enzyme activity is lost in the process.