Iodixanol (IDX) and iohexol (IHX) have been investigated as possible radiopacification agents for polymethylmethacrylate (PMMA) bone cement, to replace the currently used barium sulphate and zirconia. IDX and IHX are both water-soluble iodine-based contrast media and for the last 20 years have been used extensively in clinical diagnostic procedures such as contrast media enhanced computed tomography, angiography and urography. One of the major reasons to remove the current radiopacifying agents is their well-documented cytotoxicity and their potential to increase bone resorption. Using in vitro bone resorption assays, the effect of PMMA particles plus IDX or IHX to induce osteoclast formation and lacunar resorption on dentine slices has been investigated. These responses have been compared with the in vitro response to PMMA particles containing the conventional radiopacifying agents, that is, barium sulphate and zirconia. In parallel, the in vivo reaction, in terms of new bone formation, to particles of these materials has been tested using a bone harvest chamber in rabbit tibiae. In vitro cell culture showed that PMMA containing IHX resulted in significantly less bone resorption than PMMA containing the conventional opacifiers. In vivo testing, however, showed no significant differences between the amounts of new bone formed around cement samples containing the two iodine-based opacifying agents in particulate form, although both led to fewer inflammatory cells than particles of PMMA containing zirconia. Our results suggest that a non-ionic radiopacifier could be considered as an alternative to the conventional radiopacifying agents used in biomaterials in orthopaedic surgery.