Background: To compare nondestructive in vivo and ex vivo micro-computed tomography (μCT) and ex vivo dual-energy- X-ray-absorptiometry (DXA) in characterizing mineralized cortical and trabecular bone response to prostate cancer involving the skeleton in a mouse model. Methodology/Principal Findings: In vivo μCT was performed before and 10 weeks after implantation of human prostate cancer cells (MDA-PCa-2b) or vehicle into SCID mouse femora. After resection, femora were imaged by nondestructive ex vivo specimen μCT at three voxel sizes (31 μ, 16 μ, 8 μ) and DXA, and then sectioned for histomorphometric analysis of mineralized bone. Bone mineral density (BMD), trabecular parameters (number, TbN; separation, TbSp; thickness, TbTh) and mineralized bone volume/total bone volume (BV/TV) were compared and correlated among imaging methods and histomorphometry. Statistical tests were considered significant if P,0.05. Ten weeks post inoculation, diaphyseal BMD increased in the femur with tumor compared to the opposite femur by all modalities (p,0.005, n = 11). Diaphyseal BMD by in vivo μCT correlated with ex vivo 31 and 16 μm μCT and histomorphometry BV/TV (r = 0.91-0.94, P,0.001, n = 11). DXA BMD correlated less with bone histomorphometry (r = 0.73, P,0.001, n = 11) and DXA did not distinguish trabeculae from cortex. By in vivo and ex vivo μCT, trabecular BMD decreased (P,0.05, n = 11) as opposed to the cortex. Unlike BMD, trabecular morphologic parameters were threshold-dependent and when using ''fixed-optimal-thresholds,'' all except TbTh demonstrated trabecular loss with tumor and correlated with histomorphometry (r = 0.73-0.90, P,0.05, n = 11). Conclusions/Significance: Prostate cancer involving the skeleton can elicit a host bone response that differentially affects the cortex compared to trabeculae and that can be quantified noninvasively in vivo and nondestructively ex vivo. [ABSTRACT FROM AUTHOR]