Real-time, closed-loop intervention is an emerging experiment-control method that promises to provide invaluable new insight into cardiac electrophysiology. One example is the investigation of closed-loop feedback control of cardiac activity (e.g., alternans) as a possible method of preventing arrhythmia onset. To date, such methods have been investigated only in vitro using microelectrode systems, which are hindered by poor spatial resolution and are not well suited for atrial or ventricular tissue preparations. We have developed a system that uses optical mapping techniques and an electrical stimulator as the sensory and effector arms, respectively, of a closed-loop real-time control system. The system consists of a 2048x1 pixel linescan CCD camera that records optical signals from the tissue. Custom image processing and control software, which is implemented on top of a hard real-time operation system (RTAI Linux), processes the data and makes control decisions with a deterministic delay of <1 ms. The system is tested in two ways: (1) it is used to control, in real-time, simulated optical signals of electrical alternans, and (2) it uses precisely timed, feedback-controlled initiation of anti-tachycardia pacing to terminate reentrant arrhythmias in an arterially perfused swine right ventricle stained with voltage-sensitive fluorescent dye di-4-ANEPPS. Thus, real-time control of cardiac activity using optical mapping techniques is feasible. Such a system is attractive because it offers greater measurement resolution than the electrode-based systems with which real-time control has been used previously.