Distinguishing between the development of functional potential in antigen-specific T helper (Th) cells and the delivery of these specialized functions in vivo has been difficult to resolve. Here, we quantify the frequency of cytokine-producing cells within the primary and memory B10.BR Th cell response to pigeon cytochrome c (PCC). In vitro analysis of acquired functional potential indicated no Th1/Th2 cytokine polarity at the peak of the primary response with surprisingly little evidence for the selective preservation of interleukin (IL)-2, tumor necrosis factor (TNF)-alpha, IL-4, and interferon (IFN)-gamma potentials into the memory compartment. However, the expression of these functional potentials appears tightly regulated in vivo. The staggered appearance of primary response cytokines directly ex vivo contrasts markedly with their rapid coordinate expression in the memory response. Frequencies of IL-2-, TNF-alpha-, IFN-gamma-, and IL-10-expressing memory responders increased over their primary response counterparts, but were still markedly lower than revealed in vitro. IL-4-, IFN-gamma-, and IL-10-expressing Th cells remained at low but stable frequencies over the first 6 d of the memory response. Analysis of T cell receptor beta chain sequences of IL-4- and TNF-alpha-expressing PCC-specific Th cells provides evidence for early functional commitment among clonal progeny. These data indicate that the development of functional potential is a consequence of initial antigen experience, but delivery of specialized functions is differentially regulated in primary and memory immune responses.