We have previously reported that activation of normal murine T lymphocytes with either anti-CD3 or Con A leads to increased phosphorylation of three phosphotyrosine-containing proteins with molecular masses of 120, 80, and 40 kDa, that antibody to the alpha beta TCR heterodimer induces phosphorylation of the 120- and 80-kDa species, and that aging impairs phosphorylation of all three substrates. In our study we determine if phosphorylation of these substrates differs among T cell subsets in old and young mice. In young mice, we found that the induced levels of all three phosphorylated substrates, 10 min after addition of the mitogenic stimulus, were significantly higher in CD8 than in CD4 cells for anti-CD3 and Con A stimulation; responses to anti-TCR were also higher in CD8 than in CD4 cells for the 80 kDa, although not the 120-kDa substrate. Aging led to significant declines in the ability to phosphorylate all three substrates in the CD8 population, and also of the 80-kDa substrate in CD4 cells. Comparison of unfractionated CD4 to purified CD4 memory T cells showed that the age-dependent shift from naive to memory T cells could explain the loss with age in p80 phosphorylation, in that memory cells were significantly less able to phosphorylate p80 than were the unseparated CD4 preparations. Phosphorylation of p40 also was significantly lower in CD4 memory cells, as was phosphorylation of p120 in responses to anti-CD3. We conclude that subsets of mouse splenic T cells display different patterns of tyrosine-specific protein phosphorylation in the first 10 min after activation, and that the accumulation of memory T cells with age can account for at least some of the age-dependent decline in tyrosine protein kinase pathways.