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This book constitutes the refeered proceedings of the 21st International Conference on Information Processing in Medical Imaging, IPMI 2009, held in Williamsburg, VA, USA, in July 2009 The 26 revised full papers and 33 revised poster papers presented were carefully reviewed and selected from 150 submissions. The papers are organized in topical sections on diffusion imaging, PET imaging, image registration, functional networks, space curves, tractography, microscopy, exploratory analyses, features and detection, image guided surgery, shape analysis, motion, and segmentation and validation.