Activation of Stat5 by many cytokines implies that it cannot alone insure the specificity of the regulation of its target genes. We have evidenced a physical and functional interaction between members of two unrelated transcription factor families, Ets-1, Ets-2 and Stat5, which could contribute to the proliferative response to interleukin 2. Competition with GAS- and EBS-specific oligonucleotides and immunoassays with a set of anti-Stat and anti-Ets families revealed that the IL-2-induced Stat5-Ets complex recognizes several GAS motifs identified as target sites for activated Stat5 dimers. Coimmunoprecipitation experiments evidenced that a Stat5/Ets-1/2 complex is formed in vivo in absence of DNA. GST-pull down experiments demonstrated that the C-terminal domain of Ets-1 is sufficient for this interaction in vitro. Cotransfection experiments in Kit225 T cells resulted in cooperative transcriptional activity between both transcription factors in response to a combination of IL-2, PMA and ionomycin. A Stat5-Ets protein complex was the major inducible DNA-binding complex bound to the human IL-2rE GASd/EBSd motif in long-term proliferating normal human T cells activated by CD2 and CD28. These results suggest that the inducible Stat5-Ets protein interaction plays a role in the regulation of gene expression in response to IL-2 in human T lymphocytes.