Opioid compounds have potent analgesic and euphoric properties. They act with specific cell-membrane receptors which have been pharmacologically defined into three major classes, mu, kappa and delta. These receptors are highly regulated with respect to their gene expression, resulting in a temporally and spatially specific pattern of distribution for each receptor. To characterize the promoter sequence of the mu opioid receptor (MOR) gene, a mouse genomic DNA library was screened under high stringency with a rat MOR (MOR-1) cDNA probe and genomic sequences for the mouse MOR gene were isolated. From one genomic clone, a 2.3-kb EcoRI fragment, which hybridized to the 5'-end of the rat MOR-1 cDNA probe, was subcloned and sequenced. This fragment contains 1.3 kb of sequence upstream of the initiation codon, extends downstream through exon 1 and includes a portion of intron 1. Primer extension analysis using mouse brain poly (A)+ RNA identified a transcription initiation site 793 bp upstream from the translation start site. Chimeric constructs of mouse MOR deletion fragments fused to a luciferase reporter gene were transfected into a human neuroblastoma cell line, SK-N-SH, which constitutively expresses endogenous MOR. These transient expression studies indicated that the 0.2-kb region upstream from the transcription initiation site possesses a functional promoter, which directs the expression of the reporter gene in vitro and may possess promoter activity for the mouse MOR gene in vivo.