Cardiomyocytes are notoriously difficult to transfect using standard techniques unless viral vectors such as recombinant adenoviruses are used. Generation of recombinant adenoviruses is, however, a complex and time-consuming procedure and not possible for every DNA construct. We therefore optimized DNA/polylysine/adenovirus complexing for efficient gene transfer in neonatal rat cardiomyocytes determining the critical parameters for this method. Importantly, not only the concentration of the various components but also the method used for plasmid purification is critical for this transfection technique. Cesium-chloride-purified DNA is inferior to anion-exchange methods for this purpose possibly because of altered ionic properties. In the second part of this study, we could demonstrate silent gene transfer into cardiomyocytes applying this optimized technique to plasmids encoding luciferase or beta-galactosidase cDNAs under the control of the cytomegalovirus immediate-early promoter. Phorbol myristate acetate and/or forskolin increased the amount of beta-galactosidase positive cells up to fivefold. Luciferase activity could even be increased as much as ninefold. These results demonstrate that the cytomegalovirus promoter is not maximally active in neonatal rat cardiomyocytes under basal conditions. In fact, a large proportion of cells is silently transfected and seems to express (an) inhibitor(s) of transcription from the CMV promoter that can be overcome by stimulation of cAMP- or protein kinase C-dependent pathways.