The IL-7R controls local accessibility of joining (J) gamma gene segments in the mouse TCR gamma locus by recruiting signal transducers and activators of transcription (STAT) 5 and transcriptional coactivators to the J gamma germ line promoters and inducing histone acetylation and germ line transcription. Because STAT consensus motifs are conserved not only in the J gamma promoters but also in the TCR gamma 3' enhancer (E gamma) elements, it is possible that STAT5 interacts with and activates E gamma. To address this question, we first showed that the lysine 4 residue of histone H3 is substantially methylated at E gamma 1 and E gamma 4 elements in wild-type early thymocytes and that the levels of the methylation are reduced in IL-7R alpha chain-deficient mice. We also showed that STAT5 has potential to elevate histone acetylation of the E gamma elements in a cytokine-dependent cell line by cytokine stimulation. Next, we demonstrated that STAT5 is recruited to the STAT consensus motifs in the E gamma elements after cytokine stimulation and that transcription factors Runt-related (Runx) and c-Myb are constitutively recruited to E gamma. Furthermore, we showed that STAT5 augments basal E gamma activity controlled by Runx and c-Myb. These results suggest that STAT5 is recruited to the consensus motifs in the E gamma elements by cytokine stimulation and augments basal E gamma activity independent of Runx and c-Myb. Therefore, this study implies that the E gamma elements might be activated in two successive steps, first by Runx and c-Myb and next by STAT5.