One favored mechanism of action of glatiramer acetate (GA) in multiple sclerosis (MS) involves the induction of GA-reactive Th2 cells that are believed to enter the central nervous system and mediate bystander suppression in response to cross-reactive myelin antigens. To test this hypothesis, we examined the proliferative response and cytokine release from peripheral blood mononuclear cells (PBMCs) of 12 MS patients treated with GA, in response to 16 myelin peptides that were previously described as immunodominant or encephalitogenic and a tetanus peptide as a control antigen. Interferon-gamma (IFN-gamma) and IL-5 (markers of Th1 and Th2 responses, respectively) were assayed by enzyme-linked immunosorbent assay (ELISA). GA-stimulated PBMCs from 9 of 12 patients (75%) proliferated to one or more myelin peptides. Among the 16 peptides tested, GA-stimulated PBMCs from the majority of the patients proliferated in response to MOG(21-44). PBMCs from two thirds of the patients produced IL-5 in response to myelin peptides, while half of them produced IFN-gamma. Th1/Th0/Th2 cytokine phenotypes demonstrated that responses from 10 of 12 patients were either Th0- or Th2-biased. Responses from two patients were Th1-biased. Conversely, some myelin-specific T-cell lines (TCLs) responded to GA by proliferation (3 of 21 TCLs), IL-5 release (11 of 21 TCLs), and IFN-gamma release (3 of 21 TCLs). These results indicate that GA-reactive TCLs can respond to a spectrum of myelin peptides in a Th2-biased fashion, which is consistent with the concept of bystander suppression. Furthermore, some myelin-specific TCLs are able to recognize GA, with a tendency to produce more IL-5 than IFN-gamma, which would suggest a systemic modulatory effect of the drug.