In previous studies we demonstrated that, following activation by mitogens or alloantigens, helper T cell precursors proliferate and differentiate in vitro to produce a population of effector cells that secrete high titers of lymphokines upon restimulation . In this report, we demonstrate that a similar effector population develops in vivo following primary antigen stimulation . When restimulated with specific antigen in vitro, CD4+ T cells from mice primed 5 to 7 days previously by subcutaneous administration of keyhole limpet hemocyanin (KLH) in adjuvant, produced high levels of interleukin 2 (IL2), IL4, and IL3, and little or no interferon y (IFN-'y) or IL-5 . The effector T cells provided excellent helper activity for in vitro antibody responses of 4-hydroxy-5-iodo-nitrophenyl acetic acid-primed B cells with the production principally of the immunoglobulin Gt (IgGI) and IgM isotypes, small quantities of IgG3, and no detectable IgG2a, or IgG2b. Antigen-specific secretion of 11,2, IL-3, and ILA by in vivo effectors was detectable by 12 hours following in vitro restimulation . IFN-y and IL5 were not detected until 48 and 72 hours of culture, respectively, and low levels ofthese lymphokines were produced . Lymphokine production by primed CD4+ T cells could be induced as early as 3 days following immunization, peaked on day 5, and declined thereafter. The kinetics of in vivo appearance of effector CD4+ T cells that produce lymphokines upon restimulation in vitro were similar for each of the lymphokines examined . Mice depleted of precursor CD4+ T cells by adult thymectomy exhibited limited capacity to generate lymphokine secreting CD4+ T cells in response to primary immunization with KLH, suggesting that the majority of lymphokine producing T cells arise from short-lived and/or precursor cells. Separation of CD4+ T cells from KLH-primed mice on the basis of expression of the lymph node-specific homing receptor, MEL14, revealed that antigen-specific production of IL-2, IL-3, IL4, and IFN-y was exclusively associated with the MEL14 subset of CD4+ T cells . Separation on the basis of CD45RB expression, demonstrated that antigen-specific lymphokine production was primarily associated with the minor CD45RB population, which has been previously associated with memory activity. Our results indicate that primary in vivo immunization leads to the development ofa transient population of helper-effectors with a unique phenotype that can produce large quantities of lymphokines and mediate excellent helper activity for B cells. This population may be of critical importance for in vivo regulation of the immune response.