Abstract Background Cerebrospinal fluid (CSF) pleocytosis may be seen in asymptomatic HIV-infected individuals. This finding complicates interpretation of CSF abnormalities when such individuals are evaluated for other central nervous system infections. The goal of this study was to determine the relationship between CSF pleocytosis, central nervous system (CNS) antiretroviral penetration, adherence to antiretroviral medication regimens, neurological symptoms and performance on neuropsychological tests. Methods Clinically stable HIV-infected individuals at any peripheral blood CD4+ T cell count or any plasma viral load were asked to attend study visits at entry and every 6 months thereafter for at least one year. At each visit, they underwent a standardized neurological and medication history; neurological examination; a brief neuropsychological test battery: venipuncture; lumbar puncture; and assessment of medication adherence. Generalized estimating equations (GEE) were used to assess the relationships between CSF pleocytosis and other variables. Results CSF pleocytosis was independently and significantly related to lack of current antiretroviral use (OR 5.9, 95% CI 1.8–18.6, p = 0.003), CD4 count > 200/ul (OR 23.4, 95% CI 3.1–177.3, p = 0.002) and detectable plasma HIV RNA (OR 3.3, 95% CI 1.1–9.4, p = 0.03). At visits where antiretrovirals were used, and taking into account detectable plasma HIV RNA, an antiretroviral regimen that contained two or more agents with good CNS penetration conferred a trend toward lower odds of CSF pleocytosis (OR 0.45, 95% CI 0.18–1.12, p = 0.087). Conclusion CSF pleocytosis is a characteristic of HIV disease that varies significantly with easily identifiable clinical and laboratory features. Use of antiretroviral agents decreases the odds of pleocytosis. This association may be stronger when the regimen contains two or more agents with good CNS penetration.