T cell receptor engagement results in the reorganization of intracellular and membrane proteins at the T cell-antigen presenting cell interface forming the immunological synapse (IS), an event required for Ca²⁺ influx. KCa3.1 channels modulate Ca²⁺ signaling in activated T cells by regulating the membrane potential. Nothing is known regarding KCa3.1 membrane distribution during T cell activation. Herein we determined whether KCa3.1 translocates to the IS in human T cells using YFP-tagged KCa3.1 channels. These channels showed identical electrophysiological and pharmacological properties as wild-type channels. IS formation was induced using either anti-CD3/CD28 antibody coated beads for fixed microscopy experiments, or Epstein Barr virus-infected B cells for fixed and live cell microscopy. In fixed microscopy experiments T cells were also immunolabeled for F-actin or CD3 that served as IS formation markers. The distribution of KCa3.1 was determined with confocal and fluorescence microscopy. We found that upon T cell activation KCa3.1 channels localize with F-actin and CD3 to the IS but remain evenly distributed on the cell membrane when no stimulus is provided. Detailed imaging experiments indicated that KCa3.1 channels are recruited in the IS shortly after antigen presentation and are maintained there for at least 15-30 min. Interestingly, pre-treatment of activated T cells with the specific KCa3.1 blocker, TRAM-34, blocked Ca²⁺ influx but channel re-distribution to the IS was not prevented. These results indicate that KCa3.1 channels are a part of the signaling complex that forms at the IS upon antigen presentation.