The capacity of glatiramer acetate (GA), a random copolymer of alanine, lysine, glutamic acid, and tyrosine to stimulate primary in vitro human and murine T cell proliferation was examined. PBMCs isolated from healthy humans and relapsing remitting multiple sclerosis patients and spleen cells from inbred strains of mice, expressing different H-2 haplotypes, were used as sources of non-GA-primed lymphocytes. GA functioned as a universal Ag, inducing dose-dependent proliferation of all non-GA-primed human and murine T cell populations tested. Moreover, GA stimulated PBMCs derived ex vivo from human cord blood, strongly suggesting that GA can activate both naive and memory T cells. The human T cell proliferative responses to GA were HLA class II DR-restricted by virtue of the ability of anti-class II Ab to inhibit T cell proliferation, and the demonstration that individual GA specific human T cell clones were HLA class II DR-restricted by either restriction element but not both. Furthermore, GA-reactive T cells secreted Th0 cytokines and expressed a diverse repertoire of TCR. Limiting dilution analysis indicated that the T cell precursor frequency among the healthy human adults tested ranged from 1: 5,000 to 1: 125,000. Given that all of the T cell populations tested were isolated from non-GA-primed donors, it appears that virtually all humans and murine strains contain significant numbers of T cell populations cross-reactive with GA. These findings may explain the recent clinical finding that daily sc administration of GA ameliorates the progression of multiple sclerosis.