T hrough a combination of fluorescence microscopy and patch-clamp analysis we have identified a striking alteration in K⁺ channel expression in terminally differentiated human CCR7^–CD45RA^– effector memory T lymphocytes (T_EM). Following activation, T_EM cells expressed significantly higher levels of the voltage-gated K⁺ channel Kv1.3 and lower levels of the calcium-activated K⁺ channel IKCa1 than naive and central memory T cells (T_CM). Upon repeated in vitro antigenic stimulation, naive cells differentiated into Kv1.3^highIKCa1^low T_EM cells, and the potent Kv1.3-blocking sea anemone Stichodactyla helianthus peptide (ShK) suppressed proliferation of T_EM cells without affecting naive or T_CM lymphocytes. Thus, the Kv1.3^highIKCa1^low phenotype is a functional marker of activated T_EM lymphocytes. Activated myelin-reactive T cells from patients with MS exhibited the Kv1.3^highIKCa1^low T_EM phenotype, suggesting that they have undergone repeated stimulation during the course of disease; these cells may contribute to disease pathogenesis due to their ability to home to inflamed tissues and exhibit immediate effector function. The Kv1.3^highIKCa1^low phenotype was not seen in glutamic acid decarboxylase, insulin-peptide or ovalbumin-specific and mitogen-activated T cells from MS patients, or in myelin-specific T cells from healthy controls. Selective targeting of Kv1.3 in T_EM cells may therefore hold therapeutic promise for MS and other T cell–mediated autoimmune diseases.