We have characterized the surface phenotype and function of long-lived, Ag-specific memory CD4+ T cells generated in vivo by immunization with keyhole limpet hemocyanin (KLH). CD4+ T cells from the spleens of mice primed more than 2 mo previously with KLH, produced high levels of IL-2 and IL-3, and low levels of IL-4 and IFN-gamma in response to in vitro restimulation with specific Ag. The KLH-primed T cells mediated carrier-specific helper activity for the antibody production by NIP-primed B cells in secondary in vitro responses to NIP-KLH. Subsets of CD4+ T cells from KLH-primed mice were isolated on the basis of surface CD45RB (23G2) by magnetic separation and were examined for functional capacity in several assays of Ag-specific recall. Virtually all of the secretion of IL-2, IL-3, IL-4, and IFN-gamma in response to restimulation with Ag in vitro was associated with, and considerably enriched in, the CD45RB- subset of CD4+ T cells. Similarly, carrier-specific helper function and Ag-specific proliferation in vitro were also confined to the CD45RB-, CD4+ subset of T cells, confirming the previous association of this surface phenotype with memory Th cell activity. We also examined expression of the lymphocyte homing receptor, MEL-14 (gp90MEL), which is required for lymphocyte extravasation to peripheral lymph nodes and is present in high levels on naive T cells. MEL-14 positive and negative subsets of CD4+ T cells from long term KLH-primed mice were evaluated for Ag-specific memory function in terms of lymphokine production, Ag-induced proliferation, and helper activity. Each of these functions was associated exclusively with the MEL-14- subset of CD4+ T cells, which exhibited responses comparable to the CD45RB- subset. These data indicate that memory Th cell function in the spleen is contained within the MEL-14-, CD45RB- subset of CD4+ T cells and suggest that memory helper cells may have different patterns of recirculation from naive T cells.