A previous study from this laboratory on adoptively transferred experimental allergic encephalomyelitis (EAE) induced by myelin basic protein-responsive (MBP+) 11C-labeled lymphocytes showed that MBP+ cells entered the central nervous system (CNS) before signs, migrated through the endothelium, and remained within the perivascular space. The majority of cells effecting CNS damage were nonradiolabeled and appeared to be host-derived and non-CNS antigen specific. The present study defined the immunocytochemical and initial structural events occurring between lymphocytes and endothelial cells (EC) on CNS blood vessels during EAE induced with MBP+ lymph node cells or T-cell lines. Monoclonal antibodies against lymphocyte function-associated molecule LFA-1 and its ligand, intercellular adhesion molecule-1 (ICAM-1), and the addressin MECA-325, a marker of mouse lymph node high endothelial venules, were tested on frozen sections in combination with the avidin-biotin-complex technique. The attachment and infiltration of lymphocytes correlated with the onset of signs and the appearance in the CNS of MECA-325 and ICAM-1 on vessels with cellular infiltrates and sometimes with plump EC. The cellular infiltrates were composed largely of LFA-1+ lymphocytes. Ultrastructurally, pseudopodia from lymphocytes were seen to attach to and penetrate EC in the CNS and form small gap junction-like contacts. On the EC surface, some processes from lymphocytes made larger synapse-like contacts while others were associated with coated pits suggestive of receptor mediation. The results are in accord with specific homing and attachment of lymphocytes to the CNS vasculature being early features of the disease process in EAE and with some CNS vessels acquiring properties of lymph node elements. Understanding of the mechanisms underlying these lymphocyte/EC interactions has therapeutic import for multiple sclerosis, for which EAE is the prime model.