The basal promoter activity of the human AT₁ receptor gene was characterized using a human hepatoma cell line with a considerably high expression of AT₁, PLC-PRF-5. Four cis-acting, positively regulating elements termed AT₁PRE1 (−113 to −102 bp), AT₁PRE2 (−49 to −43 bp), AT₁PRE3 (−5 to −2 bp) and AT₁PRE4 (+44 to +50 bp) were identified. AT₁PRE2 contained a GC-box-like sequence and bound to Sp1. AT₁PRE1 contained two tandem GC-boxes and was bound to several nuclear proteins in addition to Sp1. Nuclear proteins that were bound sequence-specifically to AT₁PRE1, AT₁PRE2 and AT₁PRE4 were found in both PLC-PRF-5 cells and 8505C cells, while those bound to AT₁PRE3 were not found in 8505C cells, which showed no expression of AT₁ and almost no promoter activity for the AT₁ gene. Significant promoter activity was still observed even when AT₁PRE1, AT₁PRE2 and AT₁PRE4 were all mutated. Mutagenesis of AT₁PRE3, however, substantially inactivated promoter activity. AT₁PRE1, AT₁PRE2 and AT₁PRE4 synergistically enhanced AT₁ gene transcription promoted by AT₁PRE3. These results suggested that AT₁PRE3 is responsible for the tissue-specific expression of the human AT₁ gene, and that AT₁PRE1, AT₁PRE2 and AT₁PRE4 function as a general enhancer in liver-derived cells.