Considerable evidence indicates that transcription of the δ-opioid receptor (dor) gene is correlated with both the expression of DOR on T cells and the capacity of DOR agonists to modulate the immunological functions of the T cell. We previously reported that increased Ikaros (Ik) binding activity over an Ik-binding site at -378 to -374 (with the translation start site designated as +1) in the mouse dor promoter was required for the enhanced transcription of dor gene in phytohemagglutinin-activated EL-4 cells, a mouse T cell line that constitutively expresses DOR. In the present study, we have analyzed further the mouse dor promoter in EL-4 cells and have demonstrated that Ik-2 homodimers bind to the -378/-374 Ik-binding site and exerts a position-dependent trans-activation effect on the dor promoter. Moreover, an E box (-185 to -180) that binds upstream stimulatory factor is essential for the dor promoter activity in both resting and phytohemagglutinin-activated T cells. Furthermore, we have demonstrated that Ik-2 and upstream stimulatory factor synergize in trans-activating the dor promoter via the putative Ik-binding site and the E box, respectively.