To selectively introduce genes into the mouse myocardium, we used a recombinant adenovirus encoding a transgene composed of a cardiac-specific promoter [the proximal human brain natriuretic peptide (hBNP) promoter] coupled to a luciferase reporter gene (Ad.hBNPLuc). Activity in vitro and in vivo was compared with Ad.CMVLuc, which contained the cytomegalovirus (CMV) enhancer/promoter. We tested cell-specific and inducible regulation of Ad.hBNPLuc in vitro. Expression was higher in neonatal cardiac myocytes than in a fibroblast cell line and was induced by interleukin-1, phenylephrine, and isoproterenol in myocytes. For in vivo experiments, Ad.hBNPLuc, Ad.CMVLuc, or vehicle was injected into the left ventricular (LV) free wall of the mouse heart. In Ad.hBNPLuc-injected mice, luciferase activity was only detected in the heart. In contrast, Ad.CMVLuc-injected mice had detectable luciferase activity in all tissues examined. Our studies indicate that 1) the cardiac-specific hBNP promoter and direct cardiac injection limit expression of the transgene to the LV free wall; and 2) transgene expression in vitro is inducible and cardiac myocyte specific. Thus the use of the proximal hBNP promoter in recombinant adenoviral vectors may allow cardiac-specific and inducible expression of therapeutic genes in vivo and prevent some of the side effects of systemic adenovirus administration.