Purpose: The purpose of this study was to compare volume measurements obtained using 2 image software packages on Digital Imaging and Communications in Medicine (DICOM) images acquired from 1 multidetector computed tomography and 5 cone-beam computed tomography devices, using different protocols for physical volume measurements. Materials and Methods: Four pieces of bovine leg were prepared. Marrow was removed from 3 pieces, leaving cortical bone exposed. The resulting space of 1 piece was filled with water, another was filled with propylene glycol, and the third was left unfilled. The marrow in the fourth sample was left fully intact. Volume measurements were obtained after importing DICOM images into the Dolphin Imaging 11.95 and ITK-SNAP software programs. Data were analyzed using 3-way analysis of variance with a generalized linear model to determine the effects of voxel size, software, and content on percentage mean volume differences between tomographic protocols. A significance level of 0.05 was used. Results: The intraclass correlation coefficients for intraobserver and interobserver reliability were, respectively, 0.915 and 0.764 for the Dolphin software and 0.894 and 0.766 for the ITK-SNAP software. Three sources of statistically significant variation were identified: the interaction between software and content (P=0.001), the main effect of content (P=0.014), and the main effect of software (P=0.001). Voxel size was not associated with statistically significant differences in volume measurements. Conclusion Both content and software influenced the accuracy of volume measurements, especially when the content had gray values similar to those of the adjacent tissues.