A novel method for the isolation of leukocytes from the spinal cords of mice during chronic relapsing experimental allergic encephalomyelitis (CREAE) was developed using discontinuous density gradients. Immunostaining of these cells, together with spinal cord sections and peripheral blood leukocytes, with a panel of monoclonal antibodies enabled a detailed profile of the kinetics and cell phenotype during CREAE to be developed. Overall, the kinetics of cell accumulation within the spinal cord correlated with disease severity. The number of cells increased from an average of 5 x 10(4) in unimmunized animals to 40 x 10(4) in paralysed animals during the initial disease episode. The cell numbers rapidly declined with clinical remission (6 x 10(4) cells/cord) and again dramatically increased during clinical relapse. Low numbers of CD3+ lymphocytes (50-150 cells) were consistently isolated from normal mice. However, the number of T cells infiltrating the spinal cord increased following immunization. The numbers of T cells, macrophages, B cells, neutrophils, and Ig-bearing cells all paralleled the clinical disease course, with T cells and macrophages (showing evidence of myelin breakdown) predominating. T cells infiltrating the spinal cord generally failed to express gamma delta T cell receptors and expressed low levels of IL-2 receptors (5% of infiltrating T cells). These cells were phenotypically dissimilar to peripheral blood leukocytes isolated in parallel, with the spinal cord having a consistently higher ratio (9:1) of CD4+ to CD8+ than the peripheral blood (7:3). The low expression of MEL-14 (L-selectin) and 16a antigen (CD45RBhigh) and the higher levels of Pgp-1 (CD44) expressed by the infiltrating T cells, compared with splenocytes, suggest a preferential recruitment/retention of distinct T cell subsets, possibly memory/primed cells, into the central nervous system from the periphery during neuroimmunological disease.