Cortical bone is an anisotropic material, and its mechanical properties are determined by its composition as well as its microstructure. Mechanical properties of bone are a consequence of the proportions of, and the interactions between, mineral, collagen and water. Mid-shaft palmar cortical tissue from the equine third metacarpal bone is relatively dense and uniform with low porosity. The mainly primary osteons are aligned to within a few degrees of the long axis of the bone. Beams of compact cortical bone were prepared to examine effects of dehydration and embedding and to study contribution of collagen and mineral to nano-scale material properties. Five beams were tested: untreated (hydrated); 100% ethanol (dehydrated); or embedded in poly-methylmethacrylate (PMMA) for one normal, one decalcified, and one deproteinated bone sample. Elastic modulus was obtained by nanoindentation using spherical indenters, with the loading direction transverse  and longitudinal to the bone axis. By selectively removing water, mineral and organic components from the composite, insights into the ultrastructure of the tissue can be gained from the corresponding changes in the experimentally determined elastic moduli.