Estimates of the apparent transverse relaxation rate (R 2 * ) can be used to quantify important properties of biological tissue. Surprisingly, the mechanism of R 2 * dependence on tissue orientation is not well understood. The primary goal of this paper was to characterize orientation dependence of R 2 * in gray and white matter and relate it to independent measurements of two other susceptibility based parameters: the local Larmor frequency shift (f L ) and quantitative volume magnetic susceptibility (Δχ ). Through this comparative analysis we calculated scaling relations quantifying R 2 ′ (reversible contribution to the transverse relaxation rate from local field inhomogeneities) in a voxel given measurements of the local Larmor frequency shift. R 2 ′ is a measure of both perturber geometry and density and is related to tissue microstructure. Additionally, two methods (the Generalized Lorentzian model and iterative dipole inversion) for calculating Δχ were compared in gray and white matter. The value of Δχ derived from fitting the Generalized Lorentzian model was then connected to the observed R 2 * orientation dependence using image-registered optical density measurements from histochemical staining. Our results demonstrate that the R 2 * and f L of white and cortical gray matter are well described by a sinusoidal dependence on the orientation of the tissue and a linear dependence on the volume fraction of myelin in the tissue. In deep brain gray matter structures, where there is no obvious symmetry axis, R 2 * and f L have no orientation dependence but retain a linear dependence on tissue iron concentration and hence Δχ .MRI contrast mechanisms | grey matter | cellular architecture | relaxation times I n many neurological diseases such as multiple sclerosis, Alzheimer's, and Parkinson, and in conditions following traumatic brain injury, microstructural changes occur in gray and white matter (1-4). One method for quantifying these microstructural changes is the mapping of the effective transverse relaxation rate (R p 2 ). Along with the longitudinal relaxation rate (R 1 ) and transverse relaxation rate (R 2 ), R p 2 has been viewed as a fundamental MRI tissue parameter, affected by several factors including myelin content (5, 6), endogenous ferritin-based (Fe 3+ ) iron (7,8), tissue microstructure (6), and paramagnetic, blood deoxyhemoglobin (9). However, a number of recent studies have reported a somewhat surprising dependence of R p 2 on tissue orientation, at least in white matter (10-12). The purpose of this paper was to investigate the mechanisms that could contribute to this orientation dependence of R p 2 in both gray and white matter. Because R p 2 is influenced by magnetic field perturbations, we examined the role of local Larmor frequency shift (f L ) and quantitative magnetic susceptibility (Δχ), parameters that relate field and frequency. Through this analysis we identified unique scaling relations that relate R 2 ′ to the local Larmor frequency shift calculated after removal of macroscopic field ...