We analyzed the effects of glatiramer acetate (GA) therapy on in vitro proliferative responses and cytokine production by lymphocytes derived from multiple sclerosis patients receiving this therapy. We confirmed that lymphocytes derived from GA naïve patients show a high frequency of response when initially exposed to GA in vitro; this frequency decreased following GA therapy. The frequency of lymphocytes responding to whole MBP stimulation did not change with GA therapy. GA- and MBP-specific T cell lines generated from these patients by repeated cycles of in vitro stimulation did not cross react. Some (23%) whole MBP-reactive T cell lines did cross react with MBP peptide 83-99. The mean levels of interferon (IFN) gamma secretion and the mean ratio of IFN-gamma/IL-5 were lower for GA-reactive cell lines, derived from patients both prior to and during GA therapy, compared to MBP-reactive T cell lines. The proportion of IFN-gamma(+) cells in unfractionated lymphocyte preparations derived from the GA-treated patients did not differ from that found for healthy controls. Our findings indicate that GA-reactive T cell lines derived from GA-treated MS patients continue to show a relative Th2 cytokine bias consistent with a bystander suppressor function. GA treatment is not associated with a cytokine phenotype shift in the total T cell or MBP-reactive T cell populations.