Osteoporosis, which is correlated with low bone mass and increased bone fragility, is responsible for about 1.2 million fractures per year in the United States. We have used the three-dimensional (3-D), X-ray tomographic microscope to image the trabecular bone architecture of the proximal tibias of six Sprague—Dawley rats in vivo. Three of these rats were then ovariectomized to induce estrogen depletion, and three remained as controls. Five weeks later, the tibias were reimaged. The ovariectomized rats lost approximately 65% of their trabecular bone volume as compared with an insignificant change in the control rats. The connectivity density of the trabecular bone, as measured by the Euler characteristic, was linearly correlated with trabecular bone volume, even in the ovariectomized rats. Hypoestrogenemic bone loss manifested itself in greatly reduced connectivity and fewer trabecular elements, but not in thinning of trabeculae. The ability to microscopically image sequential changes in the 3-D architecture of trabecular bone in vivo will allow exploration of the earliest stages of hypoestrogenemic bone loss and to more rapidly test the effectiveness of new clinical treatments for this major public health problem.