Introduction: Standard care for malignant tumors arising next to a bone structure is surgical removal with safety margins, followed by external beam radiotherapy (EBRT). Complete tumor removal can result in large bone defects. A two-step bone reconstruction technique using the induced membrane (IM) technique has proven its efficacy to bridge gap nonunion. During the first step, a spacer is placed in the bone gap. The spacer then is removed and the IM around it is filled with autologous cancellous bone graft. However, the feasibility of this technique with the addition of adjuvant EBRT between the two reconstruction steps has not yet been studied. Polymethyl methacrylate (PMMA) used to be the standard spacer material for the first step. Silicone spacers could replace them owing to their good behavior when submitted to EBRT and their easier removal from the surgical site during the second step. The aim of this study was to evaluate the influence of EBRT on the histological and biochemical properties of IM induced using PMMA or silicone as spacer. Materials and methods: The analyses were performed on PMMA- or silicone-IM with and without EBRT in a 6-mm bilateral femoral defect in 32 rats. Thickness and vessel content were measured in both groups. Bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF) content in lysates of the crushed membranes were measured by enzyme immunoassay. Finally, alkaline phosphatase activity was analyzed in human bone marrow stromal cell cultures in contact with the same lysates. Results: EBRT did not change the histological structure of the cellular internal layer or the fibrous outer layer. The nature of the spacer only influenced IM thickness, PMMA-IM with external radiotherapy being significantly thicker. EBRT decreased the vascular density of IM but was less effective on VEGF/BMP2 production. In vitro, IM could have an osteoinductive potential on human bone marrow stem cells. Conclusion: EBRT did not modify the histological properties of IMs but decreased their vascular density. VEGF and BMP2 production within IMs was not affected by EBRT. Silicone spacers are able to induce membranes with similar histological characteristics to PMMA-IM.