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The Real-time Experiment Interface (RTXI) is a fast and versatile real-time biological experimentation system based on Real-Time Linux. RTXI is open source and free, can be used with an extensive range of experimentation hardware, and can be run on Linux or Windows computers (when using the Live CD). RTXI is currently used extensively for two experiment types: dynamic patch clamp and closed-loop stimulation pattern control in neural and cardiac single cell electrophysiology. RTXI includes standard plug-ins for implementing commonly used electrophysiology protocols with synchronized stimulation, event detection, and online analysis. These and other user-contributed plug-ins can be found on the website (http://www.rtxi.org).