Splenocytes from young adult or old C57BL/6NNia mice were stimulated in vitro with the anti-CD3∈ mAb, 145–2C11, in either soluble (2C11s) or plate-bound (2C11i) form. In the young group, each mode of cell activation resulted in peak DNA synthesis at ∼48 h of culture; at this time point, the old group exhibited response levels to 2C11s or 2C11i that were ∼40% of those in the young group. However, in the presence of 2C11i, splenocytes from old donors showed a delayed peak response which approached the peak levels attained in the young group. To analyze the responsiveness of the CD4+ T cell subpopulation, this cell type was isolated from spleens of young or old mice and was stimulated in vitro with 2C11s or 2C11i, in the presence or absence of added accessory cells (T cell–depleted, irradiated splenocytes). The induction of DNA synthesis by 2C11s was accessory cell dependent, and the response in the old group were markedly reduced in comparison to those in the young group. In contrast, stimulation of DNA synthesis with 2C11i was relatively accessory cell independent, resulted in higher response levels in both age groups, and lessened the disparity between age groups. The analysis of IL-2 and IL-4 secretion by stimulated CD4+ cells revealed that, in response to 2C11s and accessory cells, only IL-2 accumulation was detectable and the levels in the young group were ∼10-fold higher than the IL-2 levels in the old group. However, stimulation of CD4+ cells with 2C11i and accessory cells yielded improved IL-2 production and a detectable IL-4 response in the old group, whereas the young group exhibited a response profile similar to that induced by 2C11s. Further analysis of the IL-2, IL-4, and IFNγ mRNA levels in 2C11i-stimulated CD4+ cells revealed that old donor cells accumulated similar levels of IL-2 transcripts, but higher levels of IL-4 and IFNγ transcripts, than young donor CD4+ cells. Finally, we analyzed splenic CD4+ cells for membrane expression of four molecules—3G11, 6C10, CD45RB, and CD44—thought to demarcate CD4+ cell subsets with restricted patterns of cytokine production. The CD4+ cell fraction of individual mice contained higher percentages of cell phenotypes associated with increased IL-4:IL-2 production ratios (i.e., 3G11lo, CD45RBlo) and with increased IFNγ synthesis (i.e., CD44hi). Taken together, these data show marked alterations in the CD4+ cell subset composition in old mice, detected at the levels of subset marker expression and profiles of cytokine production. Moreover, conclusions regarding CD4+ cell competency in old donors can differ depending on the choices of stimuli and readouts for cell function in the experimental design. Therefore, age-related differences in T cell reactivity in vitro may be partially explained by the shifts in the representation of individual CD4+ subsets, each with potentially unique activation requirements and functional attributes.