Adoptive T-cell therapy with CD8(+) CTLs is often characterized by poor persistence of the transferred T cells and limited effector responses. Improved persistence and therapeutic efficacy have been noted when antigen-activated CD8(+) T cells express properties of memory cells. The current study was undertaken to more precisely characterize the development of memory-like CD8(+) T cells from short-term CTLs in vitro and upon transfer in vivo, including their antitumor activity. Ovalbumin (OVA)-specific OT-I CTLs acquired phenotypic and functional properties of memory cells 2 to 3 days later either by lowering the concentration of antigen to a level that does not support primary responses and providing a survival signal through transgenic Bcl-2 in vitro or simply by transferring early day 3 CTLs to antigen-free lymphoid-replete mice. In lymphoid-replete mice, established OVA-expressing E.G7 tumor was rejected by short-term CTLs that simultaneously acquired memory-like properties in secondary lymphoid tissues, where tumor antigen level remained low. Collectively, these data indicate that CTLs readily converted to memory-like cells upon lowering antigen to a concentration that selectively supports memory responses and suggest that such conversion predicts successful adoptive immunotherapy.