Reversible opening of the endothelial barrier remains a major obstacle when hearts are transfected via the coronary system. Our aim was to establish an experimental system permitting the continuous analysis of vascular transfer of virus in the intact heart. Isolated saline-perfused rat hearts were inverted and covered with a latex cap to collect interstitial transudate (IT) on the pericardial surface. Adenovirus (10(9) pfu/ml) was stably labeled with rhodamine fluorescent dye. Analysis of IT and coronary perfusate revealed that under baseline conditions, adenovirus in the IT reached 75% of its vascular concentration within 3 min. The nitric oxide-donors S-nitroso-N-acetyl penicillamine (SNAP) and bradykinin (BK) were the most effective substances to increase total IT volume and adenoviral interstitial concentration. Perfusion with 9% serum markedly reduced IT volume flow and delayed the SNAP/BK effect. Our findings demonstrate that SNAP and BK effectively increased coronary transfer of adenovirus suggesting that the inverted isolated heart is a suitable model to optimize vascular transfer of virus under standardized conditions.