Here, we reevaluate the effects of early termination of infection on primary T cell expansion, subsequent memory cell development, and protective immunity. Using a murine Listeria monocytogenes (LM) infection model, we found the primary expansions of both CD4(+) and CD8(+) T cells were affected even when ampicillin was given as late as 60 h postinfection (p.i.). Subsequent development of CD8(+) memory T cells was also impaired, although to a lesser extent, and only mice that received ampicillin at 24 h p.i. revealed a significant decrease in memory CD8(+) T cells. Upon rechallenge with 1 x 10(5) CFU of LM, all ampicillin-treated mice cleared LM as effectively, and they generated similar amounts of Ag-specific CD8(+) T cells as with untreated mice. However, mice that received ampicillin at 24 h p.i. lost their protective abilities when rechallenged with 7.5 x 10(5) CFU of LM. Ampicillin treatment also revealed early down-regulation of B7.1 and B7.2, but not CD40, on dendritic cells 72 h p.i. Our results have several important implications: 1) they argue against the hypothesis that brief exposure of T cells to an Ag is sufficient for full-fledged primary T cell responses and subsequent memory T cell development in vivo; 2) they suggest the existence of a reservoir of memory T cells, more than the immune system can possibly expand during secondary infection; and 3) they suggest that protective capacity is correlated with the number of preexisting memory T cells and that secondary expanding T cells play a limited role, at least in murine LM infection.