Further characterization of the 5'-flanking promoter region of the human beta1-adrenergic receptor (AR) gene was attempted. The transcription initiation sites, determined by the primer extension and the rapid amplification of the 5'-cDNA end, are multiple in a spanning about 30 nucleotides (-289 to -261 relative to the translation start site). There exist inverted CCAAT boxes, multiple binding sites for transcription factor Sp1 and AP-2 nearby transcription initiation sites, however, this region lacks a typical TATA box. In order to localize the regulatory region for the basal transcription of the human beta1-AR gene, a variety of 5'-flanking sequence/chloramphenicol acetyltransferase reporter gene fusion constructs was prepared and transiently expressed in HeLa cells. Functional analyses reveal negatively (-3813 to -2925 and -1772 to -796) as well as positively (-2925 to -1772) regulatory regions, in addition to the region (-796 to -87) being necessary for the basic expression of the human beta1-AR gene.