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MR imaging of hyperpolarized (HP) nuclei is challenging because they are typically delivered in a single dose of nonrenewable magnetization, from which the entire image must be derived. This problem can be overcome with HP 129Xe, which can be produced sufficiently rapidly to deliver in dilute form (1%) continuously and on-demand. We demonstrate a real-time in vivo delivery of HP 129Xe mixture to rats, a capability we now routinely use for setting frequency, transmitter gain, shimming, testing pulse sequences, scout imaging, and spectroscopy. Compared to images acquired using conventional fully concentrated 129Xe, real-time 129Xe images have 26-fold less signal, but clearly depict ventilation abnormalities. Real-time 129Xe MRI could be useful for time-course studies involving acute injury or response to treatment. Ultimately, real-time 129Xe MRI could be done with more highly concentrated 129Xe, which could increase the signal-to-noise ratio by 100 relative to these results to enable a new class of gas imaging applications. Magn Reson Med 60:14–20, 2008. © 2008 Wiley-Liss, Inc.