To elucidate the transcriptional regulation of the endothelin-A receptor (ET-AR) gene, we defined the cis elements in the 5'-flanking region by functional analysis. Chimeric gene constructs were prepared using serial deletion mutants of a 5-kilobase (kb) fragment of the human ET-AR 5'-flanking region and the luciferase gene and were transfected into a rat vascular smooth-muscle cell line, A7r5. Deletion of the region between -2.0 kb and -857 bases caused a twofold increase in luciferase activity, and deletion from -137 bases to -53 bases reduced the activity by 50%. These results indicate the existence of a negative regulatory element and a positive regulatory element, respectively. The gel shift assay revealed the existence of nuclear proteins in A7r5 cells that bind to the region containing the positive regulatory element. Thus, we identified cis regulatory elements and nuclear factors that regulate the expression of the ET-AR gene.