CTL killing capacity is acquired between 24 and 48 hours after stimulation. F5 Rag^−/− splenocytes were transferred by intravenous injection into C57BL/6 mice that were infected 24 hours later with recombinant vaccinia virus expressing NP366-374. In vivo cytotoxicity assays were performed using C57BL/6 splenocytes labeled with CFSE or CTO and coated with NP366-374 (R4: high concentration CFSE), PA224-233 (R3: low concentration CFSE), or medium alone (R2: CTO). Blood samples were taken 10 minutes, and 2, 4, and 18 hours following transfer of targets and the resulting lymphocytes analyzed using FACS. Spleen, abdominal lymph node, liver, and lung samples were taken at 2, 4, and 19 hours after transfer. Loss of NP366-pulsed CFSE^hi cells was attributed to antigen-specific CTL lysis. Killing capacity was assessed in mice at 0, 24, 48, and 96 hours after infection. (A) Representative FACS plots of cytotoxicity assays at 96 hours after infection are shown. (B) Specific loss of NP366-pulsed target cells at each sampling time following injection of target cells in each compartment was calculated using the formula: 1 − (% NP366 pulsed/% PA224 pulsed) × 100. Graphs represent data from at least 3 mice with standard error shown. (C) Rate of specific killing over the period between each sampling point was calculated using the formula: 1− (% NP366 pulsed at assay time point/% PA224 pulsed at assay time point)/(% NP366 pulsed at previous assay time point/% PA224 pulsed at previous assay time point) × 100. The results are plotted on a timeline from the time of infection, with the results of each assay in their relative position. Graphs represent data from at least 3 mice used in each cytotoxicity assay with standard error shown. (D) Rates of specific killing were calculated as described for control animals infected with recombinant vaccinia expressing the OVA peptide.