Viral infections induce extensive T cell proliferation in vivo. However, only a small fraction (1-5%) of the activated T cells have been shown to be virus specific leading to the prevailing notion that most of the T cell expansion represents cytokine-mediated by-stander activation and/or cross reactive stimulation of non specific cells. To re-examine this issue we quantitated antigen specific CD8 T cells during acute LCMV infection of mice using three sensitive techniques: (i) intracellular cytokine production, (ii) single cell ELISPOT and (iii) direct visualization of antigen specific CD8 T cells by staining with MHC class I tetramers + peptide. In contrast to previous estimates, we found that 50-70% of the activated CD8 T cells were LCMV specific. This represented > or = 10,000-fold increase (approximately 2 x 10(7) virus specific cells/spleen) in 8 days with the peak expansion occurring between day 3 and 5 during which period virus specific CD8 T cells had an estimated division time of approximately 8 hours. Following viral clearance, the number of antigen specific CD8 T cells dropped to 1 x 10(6) per spleen and were maintained at this level for the life of the mouse. Upon rechallenge with LCMV, memory CD8 T cells rapidly proliferated and again comprised > 50% of the total CD8 T cells. In contrast, upon challenge with a heterologous virus such as vaccinia, there was no change in the number of LCMV specific memory CTL, despite a substantial increase in the number of activated CD8 T cells. Taken together, these results show that much of the CD8 T cell expansion seen during viral infection represents antigen specific cells.