Measurement of key parameters of the microstructure of trabecular bone is critical to the study of osteoporosis and bone strength. Density based methods cannot provide this information, and give only the total amount of bone present, and not its arrangement. Magnetic resonance imaging has shown the potential to provide information related to the microarchitecture of the trabecular bone matrix. Twelve samples (8 x 8 x 8 mm3 bone cubes) were cut from sheep vertebrae such that the trabeculae ran either parallel or perpendicular to each face. Detailed measurements of the structure of these bone cubes were made by histomorphometry, and compared to R'2 and R*2 measured with a spin and gradient-echo sequence, Partially Refocused Interleaved Multiple Echo, at 1.5 Tesla. The precision of the R'2 measurement (% coefficient of variation) was 8.7+/-5.1, and 7.7+/-4.3 for R*2. Uncorrected values of R'2 and R*2 were significantly correlated to density measured by quantitative computed tomography (r = 0.87, p = 0.0005, and r = 0.90, p = 0.0002, respectively), and trabecular bone area measured by histomorphometry (r = 0.80, p = 0.002, and r = 0.83, p = 0.0008, respectively). Density correction was effected by imaging the same slice of bone in two orientations (90 degrees and 0 degrees ) to the main magnetic field. For both R'2 and R*2 there was a significant difference between measurements in the 90 degrees and 0 degrees orientations (p < 0.01). The difference between the two values was used, and termed R'2net or R*2net. The net parameters were independent of bone mass. R'2net and R*2net were significantly correlated to trabecular separation (p < 0.05) with r = -0.58 and r = -0.62, respectively. These results demonstrate the ability of magnetic resonance imaging to characterize a key measure of the trabecular microstucture. An increase in trabecular separation has important biomechanical consequences in osteoporosis. This result also strengthens the hypothesis that the sensitivity of R'2 to osteoporosis-related bone changes is due to magnetic susceptibility effects in which rapid transitions between bone and marrow create local magnetic field inhomogeneities that result in an increase in R'2 values.