The basis of antiviral protection by memory cytotoxic T lymphocytes (CTL) was investigated in vivo and in vitro using lymphocytic choriomeningitis virus (LCMV) and recombinant vaccinia viruses expressing the LCMV-glycoprotein (vacc-GP) or -nucleoprotein (vacc-NP). The widely replicating LCMV with a tendency to persist induced solid long-term protective memory. The poorly replicating vaccinia recombinant viruses revealed in the vaccinated host that the antiviral capacity of the secondary immune T cell response and the protection against lethal LCM was dependent upon the immunizing antigen and its dose. Protection against lethal choriomeningitis is less sensitive to assess memory because it depends upon high levels of CTL precursors (p) and/or on an activated state of memory CTL. In contrast, antiviral protection measured as the capacity of the primed host to reduce virus titers after challenge infection correlated with elevated CTLp frequencies after immunization with live LCMV or recombinant vaccinia virus-expressing the major LCMV epitope. CTLp frequencies were constantly increased up to 70 d for LCMV immune mice, but rapidly decreased a few weeks after immunization with low dose vaccinia recombinant virus. For example, mice primed with 2 x 10(6) plaque-forming units (PFU) of vacc-NP, or 2 x 10(2) PFU, or 2 x 10(6) PFU of vacc-GP were antivirally protected on day 7 but not after day 30 when CTLp could not be measured any longer in vitro. However, greater priming doses of vacc-NP (10(4) or 2 x 10(6) PFU) as well as LCMV (2 x 10(2) PFU) induced elevated levels of CTLp and antiviral protection for 60 d or longer. Adoptive transfer experiments of immune spleen cells into syngeneic recipients without addition of antigen demonstrated that maintenance of the antiviral protective capacity of the transferred cells depended on the presence of viral antigen. Thus, antiviral protection by memory CTL may be rather short-lived since it is based on activated T cells continuously stimulated by persisting antigen. This is best achieved by high immunizing antigen doses yielded either by widely replicating viruses or high doses of poorly replicating recombinant vaccines.