The objective of this study was to develop and validate a technique for both 3D imaging and quantification of the vascular network of bone tissue in the rat. Five month-old male Wistar rats were divided into tailsuspension (21 days) and control groups. Sixty percent barium sulfate solution was infused into the vena cava. The tibiae were evaluated in 2D and 3D before and after decalcification, using conventional microcomputerized tomography (lCT) at 10 and 5 lm resolution and synchrotron radiation (SR) lCT. The perfusion technique and tomography exhibited excellent bone vasculature imaging. Significant positive correlations were observed between 2D histomorphometric and 3D lCT vascular parameters (P < 0.05). 3DlCT discriminated significant changes of vessel structures in unloading condition: vessel number decreased by 25%, (P < 0.005), vessel separation increased by 27%, P < 0.01. SRlCT could image sinusoid clusters in bone. lCT is an accurate and reproducible technique for 3D quantitative evaluation of long bone vascularisation in the rat. Anat Rec,