Tuberculostearic acid, a structural component of Mycobacterium tuberculosis, was identified by gas chromatography/mass spectrometry with selected ion monitoring in cerebrospinal fluid (CSF) from 13 patients with proven and 8 out of 9 patients with suspected tuberculous meningitis; the negative result was in a patient whose symptoms and CSF abnormalities may have been due to systemic lupus erythematosus. Tuberculostearic acid was found in the CSF of only 1 patient out of 87 with non-tuberculous meningitis or non-infectious disorders; the single false-positive result was probably caused by intrathecal treatment with amikacin. Other aminoglycosides and antituberculous drugs did not interfere with the assay. In serial CSF samples from patients with tuberculous meningitis tuberculostearic acid was still present after 8 months of continuous supervised chemotherapy. Detection of tuberculostearic acid in CSF is a rapid, sensitive, and specific test for tuberculous meningitis, and can be used for retrospective diagnosis in patients who have been started on therapy.