Influenza A virus infection of C57BL/6 (B6) mice is characterized by prominent CD8 + T cell responses to H2D b complexed with peptides from the viral nucleoprotein (NP 366 , ASNENMETM) and acid polymerase (PA 224 , SSLENFRAYV). An in vivo cytotoxicity assay that depends on the adoptive transfer of peptide-pulsed, syngeneic targets was used in this study to quantitate the cytotoxic potential of D b NP 366 - and D b PA 224 -specific acute and memory CD8 + T cells following primary or secondary virus challenge. Both T cell populations displayed equivalent levels of in vivo effector function when comparable numbers were transferred into naive B6 hosts. Cytotoxic activity following primary infection clearly correlated with the frequency of tetramer-stained CD8 + T cells. This relationship looked, however, to be less direct following secondary exposure, partly because the numbers of CD8 + D b NP 366 + T cells were greatly in excess. However, calculating the in vivo E:T ratios indicated that in vivo lysis, like many other biological functions, is threshold dependent Furthermore, the capacity to eliminate peptide-pulsed targets was independent of the differentiation state (i.e., primary or secondary effectors) and was comparable for the two T cell specificities that were analyzed. These experiments provide insights that may be of value for adoptive immunotherapy, where careful consideration of both the activation state and the number of effector cells is required.