It has long been thought that collagen fibers within the bone matrix are deposited in an aligned pattern that channels mineral growth. If this model of bone structure is correct, both organic and inorganic phases of bone should have similar elastic anisotropy. Using an acoustic microscope, we measured longitudinal and transverse acoustic velocities of cortical specimens taken from 10 dog femurs before and after removal of either the mineral (using 10% EDTA) or collagen phases (using 7% sodium hypochlorite) and calculated longitudinal (CL) and transverse (CT) elastic coefficients. The anisotropy ratio (CL/CT) decreased significantly after demineralization (1.61 before versus 1.06 after, P<0.0001, paired t-test). However, there was no significant change after decollagenization (1.51 before versus 1.48 after, P=0.617, paired t-test). We conclude that the orientation of mineral crystals is the primary determinant of bone anisotropy, and the collagen matrix within osteonal bone has little directional orientation.