Following acute lymphocytic choriomeningitis virus (LCMV) infection, there is a potent antiviral CD8 T-cell response that eliminates the infection. This initial CD8 T-cell response is followed by a period of memory during which elevated numbers of virus-specific CD8 T cells remain in the mouse. CD4 T cells are also activated after LCMV infection, but relatively less is known about the magnitude and duration of the CD4 response. In this study, we used intracellular staining for interferon-gamma to measure both CD4 and CD8 responses in the same mice at the single cell level. After LCMV infection, there was an increase in the number of activated CD4 T cells and an associated increase in the number of virus-specific CD4 T cells. At the peak of this expansion phase, the frequency of virus-specific CD4 T cells was 1 in 20 (0.5-1.0 x 10(6) per spleen). Like the CD8 response, long-term CD4 memory could be found up to a year after the infection with frequencies of approximately 1 in 260 (0.5-1.5 x 10(5) per spleen). However, the magnitude of virus-specific CD8 T cells was greater than virus-specific CD4 T cells during all phases of the immune response (expansion, death, and memory). At day 8, there were 20- to 35-fold more virus-specific CD8 T cells than CD4 T cells. This initial difference in cell number lasted into the memory phase as there remained a ten- to 20-fold difference in the CD8 and CD4 responses. These results highlight the importance of the expansion phase in determining the size of the memory T-cell pool. In addition to the difference in the magnitude, the activation requirements of CD8 and CD4 T-cell responses were different: CD8 T responses were not affected by blockade of CD40-CD40 ligand interaction whereas CD4 responses were reduced 90%. So while there is long-term memory in both the CD8 and CD4 compartments, the rules regulating the activation of CD8 and CD4 T cells and the overall magnitude of the responses are different.