The 5'-flanking region of the mouse μ opioid receptor (MOR) gene has two promoters, referred to as distal and proximal, and the activities of each in the brain are quite different from each other. The 5'-distal promoter regulatory sequences (5'-DPRS), positioned between these two promoters, have strong inhibitory effects on the reporter gene expression driven by the MOR distal promoter. In our studies, detailed 3' deletion mapping of the 5'-DPRS narrowed down the negative cis-acting element to a 34-base pair (bp) segment (position -721 to -687). This 34-bp cis-acting element functions in both neuronal (NMB) and non-neuronal (CHO and RAW264.7) cultured cells. S1 nuclease protection assays indicated that this 34-bp cis-acting element suppresses distal promoter activity at the transcriptional level. Linker scanning mutagenesis demonstrated that nucleotides around position -721 and - 689 in the 34-bp cis-acting element are essential for the regulation of distal promoter activity. Operational characterization of the 34-bp cis- acting element in the homologous MOR distal promoter and the heterologous SV40 promoter showed that its effects are position- and promoter-dependent while being orientation-independent in both promoters. Collectively, these data suggested that this 34-bp segment is a conditional transcriptional cis- acting element that blocks mouse MOR gene expression from the distal promoter.