AbstractCMV‐specific CD8+ T cell responses in peripheral blood (PB) are characterized by a preponderance of effector and effector memory T cells. CMV‐specific central memory T cells (TCM), which are considered crucial in maintaining long‐term immunity, are rarely detectable in PB. In this study we have analyzed differentiation and function of CMV pp65‐specific CD8+ T cells in paired samples of human PB and BM using intracellular cytokine and tetramer staining. Overall frequencies of CMV pp65‐specific T cells were similar in PB compared to BM; however, CMV‐specific CD45RA–CCR7+ TCM were almost exclusively detectable in BM, which was not related to a general accumulation of TCM in BM. In vitro, CMV‐specific T cells could be more efficiently expanded from BM (median 128‐fold, n=6) than from PB (median 72‐fold, p=0.01). Taken together, these data show that the BM is a compartment harboring CMV‐specific TCM and underline the concept of the BM as a secondary immune organ. CMV specific BM‐derived TCM might be a valuable source for generating T cells for adoptive transfer.