We combined biochemical measurements with novel techniques for image analysis in the rat femur to characterize the location and nature of the defect in mineralization known to occur in growing animals after spaceflight. Concentrations of mineral and osteocalcin were low in the distal half of the diaphysis and concentrations of collagen were low with evidence of increased synthesis in the proximal half of the diaphysis of the flight bones. X-ray microtomography provided semiquantitative data in computer-generated sections of whole wet bone that indicated a longitudinal gradient of decreasing mineralization toward the distal diaphysis, similar to the chemistry results. Analysis of embedded sections by backscattered electrons in a scanning electron microscope revealed distinct patterns of mineral distribution in the proximal, central, and distal regions of the diaphysis and also showed a net reduction in mineral levels toward the distal shaft. Increases in mineral density to higher fractions in controls were less in the flight bones at all three levels, with the most distal cross-sectional area most affected. The combined results from these novel techniques identified the areas of femoral diaphysis most vulnerable to the mineralization defect associated with spaceflight and/or the stress of landing.