MR microimaging at 16.4 T with 40-μm isotropic voxels was used to investigate compartmentation of water diffusion in formalin-fixed prostate tissue. Ten tissue samples (∼ 28 mm3 each) from five organs were imaged. The mean diffusivity of epithelial, stromal, and ductal/acinar compartments was estimated by two methods: (1) manual region of interest selection and (2) Gaussian fitting of voxel diffusivity histograms. For the region of interest-method, the means of the tissue sample compartment diffusivities were significantly different (P < 0.001): 0.54 ± 0.05 μm2/ms for epithelium-containing voxels, 0.91 ± 0.17 μm2/ms for stroma, and 2.20 ± 0.04 μm2/ms for saline-filled ducts. The means from the histogram method were also significantly different (P < 0.001): 0.45 ± 0.08 μm2/ms for epithelium-containing voxels, 0.83 ± 0.16 μm2/ms for stroma, 2.21 ± 0.02 μm2/ms for duct. Estimated partial volumes of epithelial, stromal, and ductal/acinar compartments in a “tissue only” subvolume of each sample were significantly different (P < 0.02) between cancer and normal tissue for all three compartments. It is concluded that the negative correlation between apparent diffusion coefficient and cancer Gleason grade observed in vivo results from an increase of partial volume of epithelial tissue and concomitant decrease of stromal tissue and ductal space. Magn Reson Med, 2012. © 2011 Wiley Periodicals, Inc.