Abstract The mammalian ear contains three middle-ear bones called ossicles that transmit both air-conducted (AC) sound from the eardrum to the inner ear and bone-conducted (BC) vibrations of the skull to the inner ear. The functional significance of having three ossicles to transmit sound is not completely understood, yet their varied shapes, mass distributions, and articulation around two flexible joints could serve to protect the inner ear from static pressure and impulsive AC sounds presented in the ear canal, and could reduce sensitivity to potentially distracting self-generated BC vibrations caused by head movement, chewing, etc. At the same time, ossicles might also improve AC and BC hearing at low frequencies. In this study, we propose to test the role that ossicular shape, mass and mass distribution, as well as flexibility play on 3D ossicular motion and sound transmission into the cochlea for both human and elephant temporal bones in response to AC and BC stimulation under normal and modified conditions. Despite significant anatomical differences, humans and elephants exhibit very similar audiograms over their overlapping 20 Hz–11 kHz frequency range, although elephants can hear below 20 Hz and humans can hear above 11 kHz. Middle-ear bones scale with skull size, such that elephant ossicles (the largest among terrestrial mammals) are approximately seven times heavier than those of humans. Studies suggest that BC hearing is enhanced below 100 Hz using mass-loading to simulate greater ossicular mass, and our preliminary measurements on elephants suggest that their heavier ossicles should yield an order of magnitude better BC hearing than humans at low frequencies. BC hearing in elephants might also be enhanced due to what appears to be a partially fused incudo-malleolar joint. Thus, quantifying the structure–function relationships and mass loading within human versus elephant ears could improve our understanding of the possible optimizations and trade-offs within the middle ear. The immediate goal of this investigation is to quantitatively compare human and elephant ossicular-chain morphology and motion as it relates to input to the cochlea by measuring ossicular shape and mass distributions using µCT imaging; and measuring 3D ossicular motions in response to AC and BC stimulation using 3D laser Doppler vibrometry, for the normal and modified cases with added mass and reduced ossicular-joint flexibility. The motion measurements will be used to animate µCT reconstructions of the ossicles, and these results will be compared using moments of inertia (MOI) to quantify the functional implications of the inter-species structural differences and effects of modifications in terms of: 1) sound transmission from the ear canal to the cochlea, especially at lower frequencies; 2) the relative motion of the ossicles; and 3) the transmission of sound via bone conduction. The structure–function relationships revealed through this inter-species comparison ...