Naive CD4+ T cells initially transcribe both IL-4 and IFN-gamma when stimulated with either the mitogen Con A or Ag in the presence of IL-4 or IL-12 and, therefore, appear uncommitted as to the pathway of differentiation they will follow. However, when stimulated either with Con A or with Ag in the presence of IL-4, CD4+ T cells become primed to follow the Th2 differentiation pathway, and we show now that by 48 h of culture in this environment these cells extinguish IFN-gamma gene transcription. Likewise, priming in the presence of IL-12 leads to the development of Th1 cells, which switch off the expression of the IL-4 gene. To clarify the Th1 differentiation pathway, we performed ablation studies using IL-4 thymidine kinase transgenic mice. When the antiviral drug ganciclovir was added 1 day after primary stimulation in the presence of IL-12, IFN-gamma- and IL-4-producing cells were ablated. In contrast, when ganciclovir was added 2 days after primary stimulation, IL-4-producing cells, but not IFN-gamma-producing cells, were ablated. Thus, our studies show that by 48 h after activation, Th1 or Th2 cells have already become polarized to the differentiation pathway that they will follow. As the differentiation toward Th1 and Th2 effector cells proceeds, substantial amounts of IFN-gamma and IL-4 mRNA accumulate, while the mRNAs of the corresponding lineage (i.e., IFN-gamma in the case of Th2 cells, and IL-4 in the case of Th1 cells) diminish to undetectable levels. IL-4R is up-regulated during T cell differentiation by a mechanism mediated mainly by IL-4. The fact that IL-12 priming does not suppress IL-4-dependent IL-4R up-regulation shows that both IL-4 mRNA and cytokine are produced by IL-12-primed naive CD4+ T cells during differentiation into Th1 cells. Naive CD4+ T cells, therefore, begin as uncommitted cells which express both Th1 and Th2 cytokines that rapidly extinguish the expression of the inappropriate cytokine as the commitment toward the effector lineages is made.