Bone is a hierarchical material primarily composed of collagen, water, and mineral that is organized into discrete molecular, nano-, micro-, and macroscale structural components. In contrast to the structural knowledge of the collagen and mineral domains, the nanoscale porosity of bone is poorly understood. In this study, we introduce a well-established pore characterization technique, positron annihilation lifetime spectroscopy (PALS), to probe the nanoscale size and distribution of each component domain by analyzing pore sizes inherent to hydrated bone together with pores generated by successive removal of water and then organic matrix (including collagen and noncollagenous proteins) from samples of cortical bovine femur. Combining the PALS results with simulated pore size distribution (PSD) results from collagen molecule and microfibril structure, we identify pores with diameter of 0.6 nm that suggest porosity within the collagen molecule regardless of the presence of mineral and water. We find that water occupies three larger domain size regions with nominal mean diameters of 1.1, 1.9, and 4.0 nm—spaces that are hypothesized to associate with intercollagen molecular spaces, terminal segments (d-spacing) within collagen microfibrils, and interface spacing between collagen and mineral structure, respectively. Subsequent removal of the organic matrix determines a structural pore size of 5–6 nm for deproteinized bone—suggesting the average spacing between mineral lamella. An independent method to deduce the average mineral spacing from specific surface area (SSA) measurements of the deproteinized sample is presented and compared with the PALS results. Together, the combined PALS and SSA results set a range on the mean mineral lamella thickness of 4–8 nm.