In order to characterize the cellular composition of cerebrospinal fluid (CSF) in a healthy state and in the setting of chronic pleocytosis associated with HIV-1 (HIV) infection, multi-parameter flow cytometry was used to identify and quantitate cellular phenotypes in CSF derived from HIV-uninfected healthy controls and HIV-infected subjects across a spectrum of disease and treatment. CD4+ T cells were the most frequent CSF population and the CD4:CD8 ratio was significantly increased in the CSF compared to blood (p = 0.0232), suggesting preferential trafficking of CD4+ over CD8+ T cells to this compartment. In contrast, in HIV-infection, CD8+ T cells were the major cellular component of the CSF and were markedly increased compared to HIV-uninfected subjects (p<0.001). As with peripheral blood, the CSF CD4:CD8 ratio was reversed in HIV-infected subjects compared to HIV-uninfected subjects. Monocytes, B cells and NK cells were rare in the CSF in both groups, although absolute counts of CSF NK cells and B cells were significantly increased in HIV-infected subjects (p<0.05). Our studies show that T cells are the major cellular component of the CSF in HIV-infected and uninfected subjects. The CSF pleocytosis characteristic of HIV infection involves all lymphocyte subsets we measured, except for CD4+ T cells, but is comprised primarily of CD8+ T cells. The reduced proportion of CD4+ T cells in the CSF may reflect both HIV-related peripheral loss and changes in trafficking patterns in response to HIV infection in the central nervous system.