Electrophysiological studies in human myocardium are hampered by the lack of relevant in vitro models. As a new approach to study human ventricular myocytes in their natural environment, we used slice preparations of human ventricular myocardium as a new model for electrophysiological studies and analysed their pharmacological properties during long-term culture. Human myocardial tissue, which was removed during heart-valve replacement surgery, was cut into 300 mm slices under cardioplegic conditions. Slices prepared within 3 hours after surgery were either used in acute experiments or cultured for up to 28 days. Action potentials were recorded in an organ bath under physiological conditions and mRNA expression levels were determined by real-time RT PCR. Intracellular recordings of cardiomyocytes in fresh slices revealed typical characteristics of human endocardial action potential in terms of resting membrane potential (RMP: -77±2 mV), action potential amplitude (APA: 105±4 mV), max. upstroke velocity (dV/dt: 149±26 V/s), action potential duration at 20% repolarisation (APD20: 164±10 ms) and at 90% repolarisation (APD90: 384±20 ms). Excitation conduction after focal electrical stimulation was 0.35±0.04 m/s, reflecting the velocity in vivo. Treatment with dofetilide (hERG inhibition) or rilmakalim (KATP opener) changed APD90 significantly (to 151±3 % and 30±5 % respectively). Throughout the culture period mRNA expression of cardiac ion channels and exchangers (Nav1.5, Cav1.2, hERG1, NCX, Kir2.1) remained constant. Electrophysiological properties of cardiomyocytes cultured in their natural environment were well preserved after 28 days (RMP: -77±3 mV, APA: 95±3 mV, dV/dt: 110±14 V/s, APD20: 81±18 ms, APD90: 354±7 ms, conduction velocity 0.22±0.06 m/s), indicating a fully functional state of ion currents and intercellular coupling. Since responses to pharmacological manipulation of hERG and KATP were similarly reproducible, cultured slices can be considered suitable for pharmacologic assessment during the whole culture period. Human organotypic heart slices represent a new multicellular model for electrophysiological characterisation of human myocardium. Tissue slices may therefore provide a novel approach for functional research and drug development.