The passage of circulating lymphocytes into the central nervous system (CNS) during acute viral encephalitis was studied in vivo using fluorescently labeled cells inoculated into Sindbis virus (SV)-infected mice. Donor lymphocytes were detected in the brains of recipient animals when mononuclear cells were isolated from the CNS and screened by flow cytometry. The magnitude of this accumulation related to the duration of encephalitis in recipient mice and to the activation state of the inoculated cells. While Ag specificity did not influence lymphocyte entry into the inflamed CNS at any stage of infection, SV-immune cells were retained selectively within the brains of infected animals compared with cells of an irrelevant specificity. Coincident with the onset of CNS inflammation, ICAM-1 and VCAM-1 were up-regulated on cerebrovascular endothelium. Lymphocyte entry into the brains of infected animals during maximal inflammation could be inhibited by pretreating inoculated cells with Abs that blocked LFA-1, but not with those that blocked VLA-4 or down-regulated CD44. None of these reagents prevented lymphocyte entry into the brain at the onset of inflammation, suggesting that the earliest recruited cells utilize presently uncharacterized receptor-ligand interactions. These data show that the degree of existing inflammation and the activation state of circulating cells, but not their Ag specificity, influence lymphocyte recruitment into the brain during SV encephalitis. While CNS homing can be blocked with Abs against known adhesion molecules during peak inflammation, lymphocyte entry into the brain during early infection remains poorly characterized.