Splenocytes from young (3 to 4 mo) and aged (24 to 26 mo) C57BL/6 mice were stimulated with anti-CD3 epsilon mAb in vitro. At the time of peak DNA synthesis (day 2), cells from aged mice incorporated congruent to 60% less [3H]TdR than cells from young mice. This age-related defect was not attributable to gross differences in anti-CD3 does optima, response kinetics, accessory cell function, numbers of T cells cultured, CD4+:CD8+ cell ratios or surface levels of CD3 epsilon molecules. In an attempt to analyze pre-S phase events in these responses, we monitored CD4+ and CD8+ cells in splenocyte cultures for the time-dependent expression of three T cell activation markers: RL388 Ag and IL-2R and transferrin R. Parallel analyses of mean T cell size and cell cycle phase distributions were performed. Non-activated T cells from both age groups similarly expressed moderate levels of RL388 Ag, low levels of transferrin R, and undetectable levels of IL-2R. Analysis of stimulated T cells revealed, in both age groups: 1) detectable increases in expression of all three markers by 6 h of culture, and continued increases associated with blastogenesis and G1 phase transit and 2) a preferential stimulation of the CD8+ subset to a state of high level marker expression. Age group comparisons of activation marker expression over time suggested that the age-related defect reflects proportionally smaller fractions of CD4+ and CD8+ cells that respond normally, rather than a general defect in all T cells or a subset-specific defect. Finally, we found that supernatants from aged donor cell cultures stimulated with anti-CD3 contained less Il-2 than those of young controls. Addition of an IL-2 containing supernatant to aged donor cell cultures increased, but did not restore, the S phase response on day 2; however, the response on day 3 was comparable to the peak (day 2) response of young controls. These data suggest that exogenous IL-2 can improve the aged response, perhaps by expanding the fraction of normally reactive T cells.