Ets family members share a conserved DNA-binding ETS domain, and serve a variety of roles in development, differentiation and oncogenesis. Besides DNA binding, the ETS domain also participates in protein-protein interactions with other structurally unrelated transcription factors. Although this mechanism appears to confer tissue- or development stage-specific functions on individual Ets proteins, the biological significance of many of these interactions remains to be evaluated, because their molecular basis has been elusive. We previously demonstrated a direct interaction between the ETS domain of the widely expressed GABPalpha (GA-binding protein alpha) and the granulocyte inducer C/EBPalpha (CCAAT/enhancer-binding protein alpha), and suggested its involvement in co-operative transcriptional activation of myeloid-specific genes, such as human FCAR encoding FcalphaR [Fc receptor for IgA (CD89)]. By deletion analysis, we identified helix alpha3 and the beta3/beta4 region as the C/EBPalpha-interacting region. Domain-swapping of individual sub-domains with those of other Ets proteins allowed us to highlight beta-strand 3 and the subsequent loop, which when exchanged by those of Elf-1 (E74-like factor 1) reduced the ability to recruit C/EBPalpha. Further analysis identified a four-amino acid swap mutation of this region (I387L/C388A/K393Q/F395L) that reduces both physical interaction and co-operative transcriptional activation with C/EBPalpha without affecting its transactivation capacity by itself. Moreover, re-ChIP (re-chromatin immunoprecipitation) analysis demonstrated that GABPalpha recruits C/EBPalpha to the FCAR promoter, depending on these residues. The identified amino acid residues could confer the specificity of the action on the Ets proteins in diverse biological processes through mediating the recruitment of its partner factor.