Human Cytomegalovirus (CMV) reactivation remains a major source of morbidity in patients after solid organ and hematopoietic stem cell transplantation (HSCT). Adoptive T cell therapy (ACT) with CMV-specific T cells is a promising therapeutic approach for HSCT recipients, but might be counteracted by CMV's immune evasion strategies. HLA-C*07:02 is less susceptible to viral immune evasion suggesting HLA-C*07:02-restricted viral epitopes as promising targets for ACT. For a better understanding of HLA-C*07: 02-restricted CMV-specific T cells we used recently generated reversible HLA-C*07:02/IE-1 multimers (Streptamers) recognizing a CMV-derived Immediate-Early-1 (IE-1) epitope and analyzed phenotypic and functional T cell characteristics. Initially, we detected very high frequencies of HLA-C*07:02/IE-1 multimer(+) T cells (median = 11.35%), as well as robust functional responses after stimulation with IE-1 peptide (IFN gamma(+); median = 5.02%) in healthy individuals. However, MHC-multimer(+) and IFN gamma-secreting T cell frequencies showed a relatively weak correlation (r(2) = 0.77), which could be attributed to an unexpected contribution of CMV-epitope-independent KIR2DL2/3-binding of HLA-C*07:02/IE-1 multimers. Therefore, we developed a MHC-multimer double-staining approach against a cancer epitope-specific HLA-C* 07: 02 multimer to identify truly HLA-C*07:02/IE-1 epitope-specific T cells. The frequencies of these truly HLA-C*07:02/IE-1 multimer(+) T cells were still high (median = 6.86%) and correlated now strongly (r(2) = 0.96) with IFN gamma-secretion. Interestingly, HLA-C*07:02/IE-1-restricted T cells contain substantial numbers with a central memory T cell phenotype, indicating high expansion potential e.g. for ACT. In line with that, we developed a clinical enrichment protocol avoiding epitope-independent KIR-binding to make HLA-C* 07: 02/IE-1-restricted T cells available for ACT. Initial depletion of KIR-expressing CD8(+) T cells followed by HLA-C*07:02/IE-1 Streptamer positive selection using paramagnetic labeling techniques allowed to enrich successfully HL-AC* 07:02/IE-1-restricted T cells. Such specifically enriched populations of functional HL-AC* 07:02/IE-1-restricted T cells with significant central memory T cell content could become a potent source for ACT.