Previous studies have established that gene transfer into myocardial cells in vivo is detectable after direct injection of plasmid DNA. Recently, adenovirus vectors have been shown to provide an efficient method for gene transfer into a wide range of tissues. Therefore, this study sought to assess the efficiency and stability of adenovirus-mediated gene transfer into myocardium and to compare this method with that using plasmid-based gene transfer techniques. Adult rats underwent myocardial injection via a subdiaphragmatic approach. Gene transfer efficiency was compared using direct injection of an adenovirus vector encoding for the marker gene β-galactosidase (β-gal), a control adenovirus vector encoding for the cystic fibrosis transmembrane conductance regulator gene, a plasmid encoding for β-gal, or a control plasmid. Hearts infected with an adenovirus vector containing the β-gal gene showed significantly increased β-gal enzymatic activity compared with hearts injected with β-gal plasmid. Histological examination revealed that cardiac myocytes were the target of adenovirus-mediated gene transfer. A time course of gene expression showed that β-gal enzymatic activity peaked during the first week following injection. Adenovirus vectors provide an efficient but transient method for in vivo gene expression in myocardium.