Dual promoters were identified in the mouse κ-opioid receptor (KOR) gene. The distal promoter was located in the 5'-upstream region of exert 1 and the proximal promoter was located in the first intron of this gene. The transcription initiation site of the proximal promoter was mapped to the - 93rd nucleotide position from the ATG codon in a primer extension experiment. The expression of KOR mRNAs transcribed from these two promoters in mouse central nervous system and an embryonal carcinoma cell line P19 was confirmed in a ribonuclease protection assay. In non-neuronal tissues, only the transcripts initiated from the distal promoter were detected. The biological activities of these two promoters were determined in transient transfection of P19 cells with a series of reporters, each truncated at various 5'- upstream regions. It was concluded that the distal promoter was located between nucleotide positions -990 and -570, and the proximal promoter was located between nucleotide positions -330 and -93, relative to the translation initiation codon. The presence of dual promoters in the KOR gene suggested potential regulation of KOR expression by using different promoters.