This study demonstrates the feasibility of in vivo prostate diffusion tensor imaging (DTI) in human subjects. We implemented an EPI-based diffusion-weighted (DW) sequence with seven-direction diffusion gradient sensitization, and acquired DT images from six subjects using cardiac gating with a phased-array prostate surface coil operating in a linear mode. We calculated two indices to quantify diffusion anisotropy. The direction of the eigenvector corresponding to the leading eigenvalue was displayed by means of a color-coding scheme. The average diffusion values of the prostate peripheral zone (PZ) and central gland (CG) were 1.95 ± 0.08 × 10–3 mm2 s and 1.53 ± 0.34 × 10–3 mm2 s, respectively. The average fractional anisotropy (FA) values for the PZ and CG were 0.46 ± 0.04 and 0.40 ± 0.08, respectively. The diffusion ellipsoid in prostate tissue was anisotropic and approximated a prolate model, as shown in the color maps of the anisotropy. Consistent with the tissue architecture, the prostate fiber orientations were predominantly in the superior–inferior (SI) direction for both the PZ and CG. This study shows the feasibility of in vivo DTI and establishes normative DT values for six subjects. Magn Reson Med 52:530–537, 2004. © 2004 Wiley-Liss, Inc.