Adoptive transfer experimental autoimmune encephalomyelitis (AT-EAE), a disease resembling multiple sclerosis, is induced in rats by myelin basic protein (MBP)-activated CD4⁺ T lymphocytes. By patch-clamp analysis, encephalitogenic rat T cells stimulated repeatedly in vitro expressed a unique channel phenotype (“chronically activated”) with large numbers of Kv1.3 voltage-gated channels (≈1500 per cell) and small numbers of IKCa1 Ca²⁺-activated K⁺ channels (≈50–120 per cell). In contrast, resting T cells displayed 0–10 Kv1.3 and 10–20 IKCa1 channels per cell (“quiescent” phenotype), whereas T cells stimulated once or twice expressed ≈200 Kv1.3 and ≈350 IKCa1 channels per cell (“acutely activated” phenotype). Consistent with their channel phenotype, [³H]thymidine incorporation by MBP-stimulated chronically activated T cells was suppressed by the peptide ShK, a blocker of Kv1.3 and IKCa1, and by an analog (ShK-Dap²²) engineered to be highly specific for Kv1.3, but not by a selective IKCa1 blocker (TRAM-34). The combination of ShK-Dap²² and TRAM-34 enhanced the suppression of MBP-stimulated T cell proliferation. Based on these in vitro results, we assessed the efficacy of K⁺ channel blockers in AT-EAE. Specific and simultaneous blockade of the T cell channels by ShK or by a combination of ShK-Dap²² plus TRAM-34 prevented lethal AT-EAE. Blockade of Kv1.3 alone with ShK-Dap²², but not of IKCa1 with TRAM-34, was also effective. When administered after the onset of symptoms, ShK or the combination of ShK-Dap²² plus TRAM-34 greatly ameliorated the clinical course of both moderate and severe AT-EAE. We conclude that selective targeting of Kv1.3, alone or with IKCa1, may provide an effective new mode of therapy for multiple sclerosis.