BACKGROUND: First-generation, E1-deleted (deltaE1) adenovirus vectors currently used in cardiovascular gene therapy trials are limited by tissue inflammation, mainly due to immune responses to viral gene products. Recently, helper-dependent (HD; also referred to as "gutless") adenovirus vectors devoid of all viral coding sequences have been shown to cause low inflammation when injected intravenously or into skeletal muscles. However, HD vectors have not been evaluated in cardiovascular tissues. METHODS AND RESULTS: HD and deltaE1 vectors containing a cytomegalovirus-driven expression cassette for the green fluorescent protein (GFP) gene were administered intramyocardially to adult rats (n = 54). GFP expression was measured by ELISA at varying time intervals after gene transfer. HD and deltaE1 vectors were equally efficient at transducing the myocardium. Tissue inflammation was assessed by immunostaining for leukocytes and quantitative real-time RT-PCR for cytokine mRNA expression. Monocyte/macrophages, CD4(+) and CD8(+) lymphocytes infiltrating the myocardium were less abundant with HD than deltaE1 vectors. Transcripts levels for pro-inflammatory cytokines such as IL-1beta, tumor necrosis factor-alpha, and RANTES were decreased with HD vectors. However, both vectors were associated with a decline in GFP expression over time, although low-level expression was occasionally detectable 10 weeks after HD vector administration. The two vectors transduced endothelial cells in rat arteries (n = 11) with comparable efficiencies. Vascular GFP expression was not detectable at 10 weeks. CONCLUSIONS: HD vectors are as efficient as deltaE1 vectors at transducing the myocardium and vascular endothelium, while causing less myocardial inflammation. Thus, HD vectors may be superior to earlier-generation adenovirus vectors for cardiovascular gene therapy applications.