Patients with active multiple sclerosis (MS) have a selective loss of a subset of T helper cells (Th), detectable by two-color fluorescence-activated cell sorter analysis of peripheral blood lymphocytes. By using pairs of monoclonal antibodies to the T-cell subset markers CD4 (Th) and CD8 [T suppressor/cytotoxic cell (Ts)] and the common leukocyte markers Lp220 and Lp95-150, five phenotypically distinct T-cell subsets have been identified in peripheral blood: two CD4+ Th cell subsets and three CD8+ Ts cell subsets. The frequencies and absolute numbers of these five populations were measured in patients with active and inactive MS and were compared with those in healthy age-matched controls and in patients with other neurologic diseases. A high frequency of patients with active MS (80%) had a selective reduction of one Th subset (CD4+ Lp220+) compared with normal controls (P less than 0.001) or patients with inactive MS (P less than 0.001). Three patients examined sequentially had a further loss of the Lp220+ Th subset as disease activity progressed. The proportion of two Ts subsets was also abnormal in patients with active MS, but this defect was not restricted to that group. Total Th and Ts cell frequencies and Th/Ts ratios were not significantly different between patient and normal control groups. Thus, two-color analysis of T-cell subsets may be a more sensitive indicator than conventional single-marker assays of abnormal immune status in MS patients.