Cerebrospinal fluid (CSF) samples from patients with a variety of neurological disorders were assayed to determine the concentrations of tetrahydrobiopterin (BH4), the active cofactor of hydroxylases. Dihydroneopterin (NH2) and neopterin (N), which are linked with BH4 synthesis and are inflammatory biochemical markers, were also measured simultaneously in a number of patients. 5-Hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA), the main products of serotonin and dopamine breakdown, were analyzed in parallel whenever possible. As BH4 and NH2 are difficult to analyze owing to their instability, CSF samples were collected under special conditions to preserve the reduced BH4 and NH2. Liquid chromatographic assays and detection of the various substances measured also required particular precautions. BH4 concentrations were elevated in patients with neurological disorders such as syphilis and lupus-like disease and especially in an AIDS patient with neurological complications with an increased N/BH4 ratio.