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 (-408 to +100 relative to the transcription start site)] coupled to a luciferase reporter gene (Ad.hBNPLuc). Activity in vitro and in vivo was compared to Ad.CMVLuc, which contained the CMV enhancer/promoter. We first tested cell-specific and inducible regulation of Ad.hBNPLuc in vitro. Expression was 1000x higher in neonatal cardiac myocytes than in a fibroblast cell line, and was induced by interleukin-1ß, phenylephrine and isoproterenol in myocytes. When compared with Ad.CMVLuc, in vitro activity of the hBNP promoter was lower, but highly cardiac-specific. For in vivo experiments, Ad.hBNPLuc, Ad.CMVLuc or vehicle was injected into the left ventricular free wall (LV) of the mouse heart. Transgene expression was monitored by measuring luciferase activity in the LV, septum, right ventricle, atria, lung, liver, kidney, and skeletal muscle. Mice were killed from 1 to 28 days after injection, tissues removed and luciferase activity measured. Activity in the injected areas peaked at day 4, and remained above background levels for 28 days. All other regions of the heart had background luciferase activity. No other tissues from Ad.hBNPLuc-injected mice had luciferase levels above controls. In contrast, Ad.CMVLuc-injected mice had detectable luciferase activity in all tissues examined, and expression in the liver was 10% that of the heart. When Ad.CMVLuc was injected into the systemic circulation via the LV cavity, luciferase activity was detected in all tissues, and was highest in the liver. In contrast, luciferase activity in Ad.hBNPLuc-injected mice was detectable only in the heart. Our studies indicate that: 1) the cardiac-specific hBNP promoter and direct cardiac injection limit expression of the transgene to the injection site (LV); 2) transgene expression persists for at least 1 month; and 3) transgene expression in vitro is inducible and cardiac myocyte-specific. Thus using the proximal hBNP promoter in recombinant adenoviral vectors may allow cardiac-specific and inducible expression of therapeutic genes in vivo. Combining the hBNP promoter with direct myocardial injection may also prevent some of the side effects of systemic adenovirus administration.