Abstract: In a previous study we showed that a region from −182 to +10 bp in the mouse μ‐opioid receptor (MOR) promoter exhibited strong promoter activity. To identify protein‐DNA interactions in this fragment, gel shift and DNase I footprint analyses were performed using nuclear extracts from mouse brain and the human neuroblastoma cell line, SK‐N‐SH. Two regions, nucleotide (nt) −121 to −100 and nt −42 to −22, were identified as being specific protein binding sites. The protein‐DNA interaction in the nt −42 to −22 region was characterized in detail in this study. Methylation interference analysis of this region showed that nuclear protein from SK‐N‐SH cells contacted nucleotides within the sequence ATG‐CAAAT, which is a binding motif for octamer trans‐acting factors. An octamer‐1 (Oct‐1)‐specific antibody supershifted the protein‐DNA complex in a gel shift assay. A UV cross‐linking experiment showed that a nuclear protein, whose molecular weight is similar to that of the Oct‐1 factor, bound to the octamer element in the nt −42 to −22 region. Mutagenesis of four base pairs within the octamer cis‐acting element eliminated the specific protein binding in vitro. When the MOR‐luciferase reporter construct (−182 to +10 bp) with the same four base pairs mutated was transiently transfected into SK‐N‐SH cells, a 200% increase in transcriptional activity was observed. Collectively, these data suggest that Oct‐1 is binding to the octamer motif in the MOR gene and negatively modulating MOR gene expression.