IL-12 is a potent inducer of NK and cytolytic T cell activity, IFN-gamma production, and T cell proliferation, and is necessary for differentiation of naive T cells to the Th1 subset. We have previously shown that IL-12 promotes a primary Th1 response and suppresses a primary Th2 response in lymph nodes of mice primed with a model hapten-protein conjugate, 2,4,6-trinitrophenyl (TNP)-keyhole limpet hemocyanin (KLH). We have now extended these studies to determine the Th phenotype of the recall response following immunization with soluble Ag and IL-12. For these experiments, mice were primed with TNP-KLH with or without treatment with IL-12, allowed to progress beyond the primary immune response, and challenged by i.p. injection of TNP-KLH. The phenotype of the recall response was monitored by measuring ex vivo production of IFN-gamma and IL-4 in Ag-stimulated lymph node and spleen cell cultures. Titer and isotype of TNP-specific serum Abs were also evaluated. Mice primed with Ag+IL-12 developed a Th1 recall response, as detected by KLH-specific IFN-gamma production from cultured spleen cells and the presence of TNP-specific IgG2a Ab in serum. However, they also developed an Ag-specific Th2 recall response, as characterized by Ag-induced IL-4 production from spleen cells and the presence of high titers of anti-TNP IgG1 in the serum. Studies of the cytokine profile during the primary response revealed that IL-12 induced in spleen cells the capacity to express both IL-4 and IFN-gamma. CD4+ T cells are necessary for production of IL-4 in the spleens of IL-12-treated mice, and most likely account for the Th2 recall response detected in mice primed with Ag+IL-12. These results indicate that the Th1 phenotype induced by immunization with IL-12 and Ag is maintained so that a Th1 recall response is expressed upon subsequent challenge with Ag. However, immunization with IL-12 also supports the development of a Th2 recall response, indicating that the Th1-inducing effect of IL-12 in vivo is not accompanied by a long lasting suppression of Th2 development.