Brain natriuretic peptide (BNP) is a cardiac hormone constitutively expressed in the adult heart. We previously showed that the human BNP (hBNP) proximal promoter region from 127 to 40 confers myocyte-specific expression. The proximal hBNP promoter contains several putative cis elements. Here we tested whether the proximal GATA element plays a role in basal and inducible regulation of the hBNP promoter. The hBNP promoter was coupled to a luciferase reporter gene (1818hBNPLuc) and transferred into neonatal ventricular myocytes (NVM), and luciferase activity was measured as an index of hBNP promoter activity. Mutation of the putative GATA element at 85 of the hBNP promoter [1818(mGATA)hBNPLuc] reduced activity by 97%. To study transactivation of the hBNP promoter, we co-transfected 1818hBNPLuc with the GATA-4 expression vector. GATA-4 activated 1818hBNPLuc, and this effect was eliminated by mutation of the proximal GATA element. Electrophoretic mobility shift assay showed that an oligonucleotide containing the hBNP GATA motif bound to cardiomyocyte nuclear protein, which was competed for by a consensus GATA oligonucleotide but not a mutated hBNP GATA element. The -adrenergic agonist isoproterenol and its second messenger cAMP stimulated hBNP promoter activity and binding of nuclear protein to the proximal GATA element. Thus the GATA element in the proximal hBNP promoter is involved in both basal and inducible transcriptional regulation in cardiac myocytes.