Previously, we demonstrated that mouse mu-opioid receptor (MOR) gene expression is regulated by both distal and proximal promoters, with the latter playing a major role in controlling MOR transcription in the adult mouse brain. Here, we report studies of the relative usages of the mouse MOR dual promoters during murine development. We used the reverse transcription-polymerase chain reaction (RT-PCR) method, which gave results similar to those using binding assays or in situ hybridization. However, due to the greater sensitivity of RT-PCR method, we were able to detect the emergence of MOR as early as at embryonic day 8.5 (E8.5). We found that both proximal and distal promoters were active at E8.5. The proximal promoter initiated approximately two-thirds of total MOR transcripts at E8.5, with the distal promoter directing transcription of the remaining one-third. This is the greatest relative contribution of the distal promoter to MOR transcription we have observed during any time in development. Thereafter, the percentage of transcripts directed by the distal promoter gradually declined, and remained at a low but detectable level (approximately 5% of total MOR transcripts) throughout development and adulthood. Conversely, a progressive increase of the contribution of the proximal promoter to MOR transcription was observed during development, reaching its maximum in the adult. In summary, our results demonstrated the pivotal role of the proximal promoter in directing MOR transcription during murine development.