The goal of vaccination is the generation of immune memory, an immune state that permits rapid and intense recall responses to a pathogen. Considerable effort is being made to understand the nature of memory T cells. We report here that by extending the length of in vitro culture following a single restimulation with specific peptide, preparations of highly enriched, highly active antigen-specific CD8+ memory T cells could be obtained. These cultures were begun with splenocytes from mice primed by infection either with an attenuated strain of Listeria monocytogenes or vaccinia virus, both expressing the human immunodeficiency virus-1-gag gene. In the cultures, antigen-specific cytotoxic T lymphocyte (CTL) activity reached a maximum at about 9 days and thereafter fell to negligible values. Concomitant with the fall of CTL activity, however, we observed enrichment for a subset of CD11ahigh antigen-specific gag-tetramerpos CD8+ T cells. The cells showed little or no 4-hr CTL activity, but had high delayed (18-hr) CTL activity, and very high cytolytic activity after restimulation. They rapidly expressed interferon-gamma production. Their growth and survival after sorting was completely dependent on interleukin-2 or -15. As few as 5000 of the fluorescence-activated cell sorting-purified cells protected recipients against challenge 3 months after transfer. In response to the challenge, the cells repopulated lymphoid and non-lymphoid organs and showed a sizeable increase in number. The cells therefore demonstrate high protective activity for long periods of time. These cultured cells are thus a potential source of enriched natural memory T cells for reperfusion studies and in which the mechanisms that underlie the generation, differentiation and persistence of memory can be examined.