Based on the memory for the re-expression of certain cytokine genes, different subsets of Th cells have been defined. In Th type 1 (Th1) and Th2 memory lymphocytes, the genes for the cytokines interferon-gamma and interleukin (IL)-4 are imprinted for expression upon restimulation by the expression of the transcription factors T-bet and GATA-3, respectively, and epigenetic modification of the cytokine genes. In Th17 cells, IL-17 expression is dependent on the transcription factors RORgammat and RORalpha. Here, we analyze the stability and plasticity of IL-17 memory in Th17 cells. We have developed a cytometric IL-17 secretion assay for the isolation of viable Th cells secreting IL-17. For Th17 cells generated in vitro, IL-17 expression itself is dependent on continued TGF-beta/IL-6 or IL-23 signaling and is blocked by interferon-gamma and IL-4 signaling. In response to IL-12 and IL-4, in vitro generated Th17 cells are converted into Th1 or Th2 cells, respectively. Th17 cells isolated ex vivo, however, maintain their IL-17 memory upon subsequent in vitro culture, even in the absence of IL-23. Their cytokine memory is not regulated by IL-12 or IL-4. Th17 cells generated in vivo are a stable and distinct lineage of Th cell differentiation.