Brain natriuretic peptide (BNP) is a cardiac hormone constitutively expressed in the adult heart. To identify the cis-acting elements involved in regulation of the human BNP gene, we subcloned the full-length promoter (−1818 to +100) and deletions thereof upstream from a luciferase reporter gene and transiently transfected them into primary cultures of neonatal rat atrial and ventricular myocytes and myocardial fibroblasts. Luciferase activity of the full-length construct was higher in ventricular (39 064±8488 relative light units, n=11) and atrial (11 225±1907, n=17) myocytes than myocardial fibroblasts (329±113, n=5). Maximal promoter activity in ventricular and atrial myocytes was maintained by sequences positioned between −1818 and −1283 relative to the transcription start site. Deletion to −1175 resulted in a decrease, whereas further deletion to −500 effected an increase in reporter activity in both cell types. In ventricular and atrial myocytes, deletion from −500 to −40 reduced luciferase activity 20-fold and 2-fold, respectively, whereas in myocardial fibroblasts, deletion to −40 upregulated the BNP promoter 2-fold. Of note, deleting 16 bp between −127 and −111 reduced luciferase activity 7-fold and 4-fold in ventricular and atrial myocytes, respectively, but had essentially no effect on luciferase activity in fibroblasts. Placement of sequences lying between −127 and −40 upstream from a heterologous thymidine kinase promoter resulted in reporter expression that was 7.4-fold greater than the vector alone in ventricular myocytes, approximately 2-fold greater in atrial myocytes, and equivalent to the vector alone in fibroblasts. For study of activity of the human BNP promoter in adult myocytes, either 408 or 97 bp of 5′ flanking sequence coupled to the luciferase reporter gene was injected into the apex of adult male Sprague-Dawley rat hearts. After 7 days, luciferase activity in the injected myocardium was 9.8-fold higher for the longer construct (70 683±14 744 versus 7223±3920, n=4, P<.01), consistent with our in vitro data. These data indicate that (1) the full-length human BNP promoter is more active in ventricular versus atrial myocytes and essentially inactive in fibroblasts, (2) the distal BNP promoter contains both positive and negative regulatory elements, (3) a region of the proximal BNP promoter located between −127 and −40 confers tissue specificity, and (4) the BNP promoter is active after injection into the adult rat heart.