We cloned the hOPN gene and its 5' upstream region, and analyzed its exon-intron structure and potential regulatory sequences of the promoter region in comparison with those of mouse and porcine homologues. The hOPN gene consists of 7 exons that are similar to those of the mouse gene, although the hOPN gene is longer than the mouse homologue. This difference is attributable to an insertion of about 1750 bp immediately before exon 4 in the hOPN gene. A region of approximately 285 bp immediately upstream of the hOPN transcription initiation site was highly conserved and contained a number of potential cis regulatory consensus sequences. CAT analysis using SCC-3 cells demonstrated that nucleotides at positions -439 to -270, -124 to -80, and -55 to -39 contained cis-acting enhancing elements, in which the -124 to -80 element was much more active than the others. Deletion of the sequences between -474 and -270 localized the cis elements to the sequence at position -439 to -410, whereas the deletion between -124 to -80 localized it to -124 to -115, and -94 to -80 (data not shown). Gel shift analysis using synthesized double-stranded oligonucleotides corresponding to the 30 bp at position -439 to -410 (data not shown), and 10 and 15 bp regions at positions -124 to -115 and -94 to -80, respectively, as probes revealed that each probe formed one or two bands complexed with a nuclear protein prepared from SCC-3 cells.(ABSTRACT TRUNCATED AT 250 WORDS)