External mechanical loading of cells aligns cytoskeletal stress fibres in the direction of principle strains and localises paxillin to the mechanosensing region. If the osteocyte cell body can indeed directly sense matrix strains, then cytoskeletal alignment and distribution of paxillin in osteocytes in situ will bear alignment to the different mechanical loading patterns in fibulae and calvariae. We used confocal microscopy to visualise the immunofluorescence-labelled actin cytoskeleton in viable osteocytes and paxillin distribution in fixated osteocytes in situ. In fibular osteocyte cell bodies, actin cytoskeleton and nuclei were elongated and aligned parallel to the principal (longitudinal) mechanical loading direction. Paxillin was localised to the 'poles' of elongated osteocyte cell bodies. In calvarial osteocyte cell bodies, actin cytoskeleton and nuclei were relatively more round. Paxillin was distributed evenly in the osteocyte cell bodies. Thus in osteocyte cell bodies in situ, the external mechanical loading pattern likely determines the orientation of the actin cytoskeleton, and focal adhesions mediate direct mechanosensation of matrix strains. (C) 2008 Published by Elsevier Inc.