Lymphocyte function associated antigen-3 (LFA-3) is a cell surface glycoprotein involved in antigen independent T-cell activation and proliferation. The expression and function of LFA-3 at the blood-brain barrier were studied in an in vitro model consisting of primary cultures of human brain microvessel endothelial cells (HBMEC). Surface expression of LFA-3 was detected by immunogold silver staining and the presence of RNA by reverse transcriptase-polymerase chain reaction (RT-PCR). Unstimulated HBMEC in primary culture constitutively express LFA-3 on their surface. Expression is only marginally upregulated following stimulation with tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma (IFN-gamma). Similarly, LFA-3 RNA is present constitutively in unstimulated HBMEC with minimal increase after co-incubation with TNF-alpha and IFN-gamma. The function of LFA-3 as a costimulatory molecule on HBMEC was investigated by incubating purified CD4+ T-lymphocytes with resting or IFN-gamma treated HBMEC monolayers. Proliferation of alpha-CD3 activated CD4+ T-cells was significantly increased upon incubation with resting or activated endothelial cells. Monoclonal antibodies to LFA-3 consistently blocked the proliferative response by 64-76%. The ability of the cerebral endothelium to express LFA-3 and provide secondary signals for T-cell proliferation suggests that cerebral EC may be important in the initiation of inflammatory responses in the human central nervous system.