In humans, the pathways of memory T-cell differentiation remain poorly defined. Recently, adoptive cell transfer (ACT) of tumor-reactive T lymphocytes to metastatic melanoma patients after nonmyeloablative chemotherapy has resulted in persistence of functional, tumor-reactive lymphocytes, regression of disease, and induction of melanocyte-directed autoimmunity in some responding patients. In the current study, longitudinal phenotypic analysis was performed on melanoma antigen-specific CD8⁺ T cells during their transition from in vitro cultured effector cells to long-term persistent memory cells following ACT to 6 responding patients. Tumor-reactive T cells used for therapy were generally late-stage effector cells with a CD27^Lo CD28^Lo CD45RA^- CD62 ligand^- (CD62L^-) CC chemokine receptor 7^- (CCR7^-) interleukin-7 receptor α^Lo (IL-7Rα^Lo) phenotype. After transfer, rapid up-regulation and continued expression of IL-7Rα in vivo suggested an important role for IL-7R in immediate and long-term T-cell survival. Although the tumor antigen-specific T-cell population contracted between 1 and 4 weeks after transfer, stable numbers of CD27⁺ CD28⁺ tumor-reactive T cells were maintained, demonstrating their contribution to the development of long-term, melanoma-reactive memory CD8⁺ T cells in vivo. At 2 months after transfer, melanoma-reactive T cells persisted at high levels and displayed an effector memory phenotype, including a CD27⁺ CD28⁺ CD62L^- CCR7^- profile, which may explain in part their ability to mediate tumor destruction. (Blood. 2005;105:241-250)