The major function of cytotoxic T lymphocytes (CTLs) is to eliminate pathogen‐infected and tumorigenic cells. This is mediated mainly through the exocytosis of lytic granules (LGs) containing cytotoxic components, such as perforin and granzymes at the immunological synapse (IS). The soluble NSF attachment receptor (SNARE) protein isoforms are well known to be required for vesicle exocytosis in neuronal synapses, but their potential function in CTLs is only partly understood. Here, we examined the expression of SNARE proteins before and after the activation of primary human CD8⁺ T cells and determined their co‐localization with LGs and CD3 after IS formation with target cells. We found that several key SNARE proteins in neuronal cells were not expressed in CTLs, such as syntaxin1B2 and SNAP‐25. Vti1b, Stx8 and Stx16 had the highest degrees of co‐localization with LGs while Stx3, Stx4, Stx6, Stx7, Stx8, Stx13, Vti1b, VAMP3 and VAMP4 co‐localized with CD3. Our data provide the first complete expression profile and localization of SNAREs in primary human CD8⁺ T cells, laying the groundwork for further understanding their potential role in T‐cell function.