Establishing the diagnosis of neurosyphilis may be particularly difficult in human immunodeficiency virus type 1 (HIV)-infected persons. Polymerase chain reaction (PCR) was used to detect Treponema pallidum DNA in cerebrospinal fluid (CSF) from 81 HIV-infected patients. On the basis of reactive serum and CSF-VDRL tests, 2 patients were diagnosed as having neurosyphilis. T. pallidum DNA was not consistently detected in any sample, even when the CSF-VDRL was reactive. CSF pleocytosis, elevated protein, or depressed glucose concentration was not significantly associated with a history of exposure to or infection with T. pallidum. On the basis of results of routine CSF measurements and T. pallidum PCR results, no evidence was found for undiagnosed neurosyphilis in HIV-infected patients. T. pallidum DNA PCR on CSF did not provide more information than conventional CSF analysis. Further study is needed to determine the utility of this test in the diagnosis and treatment of neurosyphilis.