The molecular basis for changes in cytokine expression during T helper (Th) cell subset differentiation is not well understood. We have characterized transcriptional events related to cytokine gene expression in populations of naive T cell receptor-transgenic T cells as they are driven in vitro toward Th1 or Th2 phenotypes by interleukin (IL)-12 or IL-4 treatment, respectively. Quantitative reverse transcriptase-polymerase chain reaction analysis of cytokine transcripts indicates that interferon (IFN) gamma, IL-4, and IL-2 mRNA are expressed with distinct kinetics after naive T cells are stimulated with antigen and either IL-4 or IL-12. IFN-gamma mRNA appears as early as 6 h in IL-12-treated cultures, IL-4 appears only after 48 h in IL-4-treated cultures, and IL-2 is equivalently expressed in both types of cultures. Analyses were performed to determine if there were any differences in activation of IL-2 or IL-4 transcription factors that accompanied Th1 versus Th2 differentiation. These studies demonstrated that signal transducer and activator of transcription 6 (STAT6) binds to a sequence in the IL-4 promoter and that this STAT6-binding site can support IL-4-dependent transcription of a linked heterologous promoter. Prolonged activation of STAT6 is characteristic of populations undergoing Th2 differentiation. Furthermore, STAT6 is activated in an autocrine manner when differentiated Th2 populations are stimulated by antigen receptor ligation. Th1 populations derived from IL-12 plus antigen treatment of naive T cells remain responsive to IL-4 as indicated by induction of STAT6 and IL-4 mRNA. These data indicate that Th1 and Th2 differentiation represents the combination of different, apparently independently regulated transcriptional events. Furthermore, among transcription factors that bind to the IL-4 or IL-2 promoters, STAT6 is the one whose activation distinguishes Th2 versus Th1 development.