The aim of the present study was to assess the reproducibility and accuracy of measurements done on excised rat bone with three different generations of densitometers: Hologic QDR2000 pencil beam, Hologic QDR4500 fan beam, and Lunar PIXImus cone beam. The coefficients of variation for repeated measurements of bone mineral content (BMC) were 0.62 and 0.85% for pencil beam, 1.73 and 3.59% for fan beam, and 0.70 and 1.52% for cone beam for femur and tibia, respectively. BMC and ash weight were linearly correlated: 0.998 for pencil, 0.984 for fan, and 0.995 for cone beam. However, the three densitometers overestimated BMC by 10.9, 12.6, and 3.1%, respectively, and the overestimation was found to be dependent on the net BMC. The highest coefficient of correlation was found between BMC measurements from pencil and cone beam (r = 0.995). Data from cone-beam DXA were, respectively, 8.8 and 9.2% lower than those from penciland fan-beam DXA. We conclude that the three DXA instruments precisely and accurately measure BMC in excised rat bone; however, DXA overestimates BMC with a dependence on the bone ash weight. This dependence was less pronounced with the cone-beam technology.