Diffusion tensor magnetic resonance imaging provides structural information about nerve fiber tissue. The first eigenvector of the diffusion tensor is aligned with the nerve fibers, i.e., longitudinally in the spinal cord. The underlying hypothesis of this study is that the presence of collateral nerve fibers running orthogonal to the longitudinal fibers results in an orderly arrangement of the second eigenvectors. Magnetic resonance diffusion tensor scans were performed with line scan diffusion imaging on a clinical MR scanner. Axial sections were scanned in a human cervical spinal cord specimen at 625 microm resolution and the cervical spinal cord of four normal volunteers at 1250 microm resolution. The spinal cord specimen was fixed and stained for later light microscopy of the collateral fiber architecture at 0.53 microm resolution. Diffusion measured by MR was found to be anisotropic for both white and gray matter areas of the spinal cord specimen; the average fractional anisotropy (FA) was 0.63 +/- 0.09 (diffusion eigenvalues lambda1 0.38 +/- 0.05 micros/mm2, lambda2 0.14 +/- 0.03 micros/mm2, lambda3 0.10 +/- 0.03 micros/mm2) in white matter and 0.27 +/- 0.04 (lambda1 0.36 +/- 0.04 micros/mm2, lambda2 0.28 +/- 0.03 micros/mm2, lambda3 0.21 +/- 0.04 micros/mm2 in gray matter. The normal-volunteer FA values were similar, i.e., 0.66 +/- 0.04 (lambda1 1.66 +/- 0.14 micros/mm2, lambda2 0.55 +/- 0.02 micros/mm2, lambda3 0.40 +/- 0.01 micros/mm2) in white matter and 0.35 +/- 0.03 (lambda1 1.14 +/- 0.07 micros/mm2, lambda2 0.70 +/- 0.03 micros/mm2, lambda3 0.58 +/- 0.02 micros/mm2) in gray matter. The first eigenvector pointed, as expected, in the longitudinal direction. The second eigenvector directions exhibited a striking arrangement, consistent with the distribution of interconnecting collateral nerve fibers discerned on the histology section. This finding was confirmed for the specimen by quantitative pixel-wise comparison of second eigenvector directions and collateral fiber directions assessed on light microscopy image data. Diffusion tensor MRI can reveal non-invasively and in great detail the intricate fiber architecture of the human spinal cord.