An experimental in vitro model of human adult myocardium would be highly desirable. Here, we present electrophysiological and mechanical as well as structural characteristics of slice preparations from human ventricular myocardium, and their preservation in long-term culture. Myocardial tissue specimens were obtained during heart surgery, and samples were cut into 300mm thick slices within 3 hours. Slices were either used immediately or after organotypic culture for up to 28 days. Isometric force development under electrical stimulation (1 Hz) was determined acutely or after prolonged culturing periods. Fresh slices showed high viability and preservation of contractility, as shown by force measurements. Contractions of fresh slices showed a distinct preload dependency and reached optimum forces of 8 mN, corresponding to a tension of 5.3 mN/mm². Isoproterenol and calcium raised the developed force in a concentration dependent manner (EC₅₀ of 0.3 mM and 5 mM, respectively). Isoproterenol increased contractility up to 2.0 fold. Cardiomyocytes stayed viable in tissue culture for up to 28 days. Messenger RNA expression of myocyte specific genes (SERCA2, connexin 43, titin, phospholamban) and of cardiac ion channels and transporters (Ca_v 1.2, K_v 4.3, hERG1, NCX, Na⁺/K⁺-ATPase) remained constant throughout the culture period. Transcription of sarcomeric components (myosin light chain 2, a-actin 1) was down-regulated within the first day in culture, and maintained a new steady-state throughout the culture period. This altered differentiation was paralleled by a partial loss of linear sarcomer organization that also reduced the maximum developed force of contraction. However the inotropic response of cultured slices to ß-adrenergic and calcium stimulation was maintained. Accordingly, isoproterenol stimulation evoked a 2.0 fold increase in contraction force even after 21 days of culture. Our results show that human organotypic heart slices represent a suitable new model for functional research and drug testing.