PURPOSE The nature of the remarkable phase contrast in high-field gradient echo MRI studies of human brain is a subject of intense debates. The generalized Lorentzian approach (He and Yablonskiy, Proc Natl Acad Sci USA 2009;106:13558-13563) provides an explanation for the anisotropy of phase contrast, the near absence of phase contrast between white matter and cerebrospinal fluid, and changes of phase contrast in multiple sclerosis. In this study, we experimentally validate the generalized Lorentzian approach. THEORY AND METHODS The Generalized Lorentzian Approach suggests that the local contribution to frequency shifts in white matter does not depend on the average tissue magnetic susceptibility (as suggested by Lorentzian sphere approximation), but on the distribution and symmetry of magnetic susceptibility inclusions at the cellular level. We use ex vivo rat optic nerve as a model system of highly organized cellular structure containing longitudinally arranged myelin and neurofilaments. The nerve's cylindrical shape allowed accurate measurement of its magnetic susceptibility and local frequency shifts. RESULTS We found that the volume magnetic susceptibility difference between nerve and water is -0.116 ppm, and the magnetic susceptibilities of longitudinal components are -0.043 ppm in fresh nerve, and -0.020 ppm in fixed nerve. CONCLUSION The frequency shift observed in the optic nerve as a representative of white matter is consistent with generalized Lorentzian approach but inconsistent with Lorentzian sphere approximation.