Previously, we identified a minimum core promoter of the mouse δ-opioid receptor (DOR) gene. The DOR promoter contains an E-box that binds upstream stimulatory factor and is crucial for the DOR promoter activity in NS20Y cells, a mouse neuronal cell line that constitutively expresses DOR. In the present study, we further analyzed the DOR promoter in NS20Y cells and have demonstrated that transcription factor Ets-1 binds to an Ets-1-binding site overlapping the E-box and trans-activates the DOR promoter by synergizing with upstream stimulatory factor in specific DNA binding. In addition, the Ets-1 DNA-binding domain is sufficient to play the functional role of Ets-1 in trans-activating the DOR promoter. Furthermore, through in vivo cross-linking assays and Northern blot analyses, we have demonstrated that Ets-1 binds to the DOR promoter in the neonatal mouse brain and that overexpressed Ets-1 can significantly enhance the expression of DOR mRNA in primary neonatal mouse neuronal cells. Collectively, our data suggest that Ets-1 functions as a trans-activator of the DOR promoter in the neonatal mouse brain and thus may contribute to the development of the mouse brain DOR system.