Previous studies of reentrant arrhythmias in the heart have been performed in computer models and tissue experiments. We hypothesized that confluent monolayers of cardiac cells can provide a simple, controlled, and reproducible experimental model of reentry. Neonatal rat ventricular cells were cultured on 22-mm-diameter coverslips and stained with the voltage-sensitive dye RH-237. Recordings of transmembrane potentials were obtained from 61 sites with the use of a contact fluorescence imaging system. An electrical field stimulus, followed by a point stimulus, induced 39 episodes of sustained reentry and 21 episodes of nonsustained reentry. Sustained reentry consisted of single-loop (n ϭ 18 monolayers) or figure-of-eight (n ϭ 4) patterns. The cycle length, action potential duration at 80% repolarization, and conduction velocity were (in means Ϯ SE) 358 Ϯ 33 ms, 118 Ϯ 12 ms, and 12.9 Ϯ 1.0 cm/s for single loop and 311 Ϯ 78 ms, 137 Ϯ 18 ms, and 7.8 Ϯ 1.3 cm/s for figure-of-eight, respectively. Electrical termination by 6-to 13-V/cm field pulses or 15-to 20-V point stimuli was successful in 60% of the attempts. In summary, highly stable reentry can be induced, sustained for extensive periods of time, and electrically terminated in monolayers of cultured neonatal rat cardiac myocytes. arrhythmia; cardiac electrophysiology; voltage-sensitive dye; optical mapping UNIDIRECTIONAL CONDUCTION block and formation of wave breaks during propagation in heart muscle can result in self-sustained propagation of electrical and mechanical activity, in which an activation wave front can reenter the same area after propagating around a fixed anatomic or functional obstacle (9). Functional reentry is generally accepted to be the major mechanism underlying many monomorphic and polymorphic tachycardias, which can evolve into irregular electrical activity such as atrial or ventricular flutter and fibrillation. Although the initiation and cardioversion of functional reentry have been subjects of numerous theoretical and experimental studies (2-4, 12, 23, 24, 27, 31, 33), there still exists a need for the development of new model systems that can aid in the understanding and treatment of these life-threatening arrhythmias.The use of cultured monolayers of cardiac cells as a simplified model for the study of functional cardiac electrophysiology offers many advantages, including: 1) control of the cell microenvironment, 2) elimination of excitation-contraction decouplers that are used for optical mapping but may alter the electrophysiological properties of the cells (17, 18), 3) removal of large scale tissue heterogeneities such as blood vessels, connective tissue, or rotational anisotropy, and 4) the certainty that the electrophysiological signals are produced from a known layer of cells, thus enabling a one-to-one correspondence with two-dimensional computer simulations and nonlinear dynamic theory. Currently, there is a gap between the computer simulations, which assume an ideal homogeneous excitable media, and tissue experiments, where ...