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The nuclear receptor COUP-TFII was recently shown to bind to the promoter of the epsilon- and gamma-globin genes and was identified as the nuclear factor NF-E3. Transgenic experiments and genetic evidence from humans affected with hereditary persistence of fetal hemoglobin suggest that NF-E3 may be a repressor of adult epsilon and gamma expression. We show that, on the epsilon-promoter, recombinant COUP-TFII binds to two sites, the more downstream of which overlaps with an NF-Y binding CCAAT box. Binding occurs efficiently to either the 5' or the 3' COUP-TFII site but not to both sites simultaneously. However, adding recombinant NF-Y induces the formation of a stable COUP-TFII.NF-Y-promoter complex at concentrations of COUP-TFII that would not give significant binding in the absence of NF-Y. Mutations of the promoter indicate that COUP-TFII cooperates with NF-Y when bound to the 5' site, whereas binding at the 3' site is mutually exclusive. Likewise, in the gamma-promoter, COUP-TFII binds to a site overlapping the distal member of a duplicated CCAAT box, competing with NF-Y binding. Transfections in K562 cells show that both the mutation of the 5' COUP-TFII or of the NF-Y site on the epsilon-promoter decrease the activity of a luciferase reporter; the mutation of the 3' COUP-TFII site has little effect. These results, together with transgenic experiments suggesting a repressive activity of COUP-TFII on the epsilon-promoter and the observation that, on the 3' site, COUP-TFII and NF-Y binding is mutually exclusive, suggest that COUP-TFII may exert different effects on epsilon transcription depending on whether it binds to the 5' or to the 3' site. At the 5' site, COUP-TFII might cooperate with NF-Y, forming a stable complex, and stimulate transcription; at the 3' site, COUP-TFII might compete for binding with NF-Y and, directly or indirectly, decrease gene activity.