Three color flow cytometry was used to analyze immunoregulatory lymphocyte subsets in peripheral blood (PB) and cerebrospinal fluid (CSF) of 21 patients with multiple sclerosis (MS) and 15 age-matched healthy control subjects. Two cell surface antigens associated with T lymphocyte memory and activation, CD45R0 and CD29, were analyzed on the CD4+ and CD8+ subpopulations, respectively. A selective decrease in the expression of the CD45R0 isoform among CD8+ cells was noted in both PB (p < 0.005) and CSF (p > 0.0001) of patients with MS as compared with the control group while the expression of CD29 did not differ between the groups. These changes could indicate a defective differentiation into mature memory CD8+ T lymphocytes in patients with MS. Furthermore, the CD3+CD16/56+ T lymphocyte subset capable of mediating NK cell-like activities was investigated. Although this cell population is quantitatively small, a significant reduction of the proportion of this cell type was detected in both BP and CSF of the MS group compared with the controls (p < 0.01 and p > 0.001, respectively). Further studies are needed to establish the role of these observations in the pathogenesis of MS.