New method of qualitative and quantitative analysis of nucleotides in human cerebrospinal fluid (CSF), based on the combination of extraction of purines and pyrimidines to the solid phase (SPE) and high-performance liquid chromatography (HPLC), was proposed. Use of SPE and lyophilization of samples allowed for the first time to detect the presence of di- and triphosphonucleotides in human CSF. Concentration of those compounds varied from 0.003 to 5.0 microM. Differences in the nucleotide mixture composition in human CSF detected with the new method are coupled with the neurological disorders and might be a basis for an efficient diagnostic tool.