Low volume and few cells have hampered the use of flow cytometry for studying cerebrospinal fluid (CSF) in routine clinical practice, although information about the cellular phenotypes present in this type of sample is of great value in many diseases. We developed a novel flow cytometric strategy capable of identifying total CSF T lymphocytes and the CD4+ subset, even in CSF samples with as few as 1 leukocyte per 3 microL of sample. We also showed that identification of CD8+ T cells could be achieved in most samples, while B lymphocytes are detectable only in samples with more than 5 cells per microliter. These findings demonstrate the reliability of this method to improve the diagnostic accuracy of classic cytologic studies in many neurologic disorders.