Glatiramer acetate (GA) is believed to induce GA-reactive T cells that secrete anti-inflammatory cytokines at the site of inflammation in multiple sclerosis (MS). However, GA-reactive T cells have not been established from the intrathecal compartment of MS patients, and intrathecal T cells may differ from T cells in blood. Here, we compared the phenotype of GA-reactive T cells from the cerebrospinal fluid (CSF) and blood of five MS patients treated with GA for 3-36 months, and in three of these patients also before treatment. From the CSF of these patients, all 22 T cell lines generated before and all 38 T cell lines generated during treatment were GA-reactive. GA treatment induced a more pronounced anti-inflammatory profile of GA-reactive T cell lines from CSF than from blood. While GA-reactive T cell clones from CSF were restricted by either human leukocyte antigen (HLA) -DR or HLA-DP, only HLA-DR restricted GA-reactive T cell clones were detected in blood. No cross reactivity with myelin proteins was detected in GA-reactive T cell lines or clones from CSF. These results suggest that a selected subset of GA-reactive T cells are present in the intrathecal compartment, and support an anti-inflammatory mechanism of action for GA.