Bone is one of the most common metastatic target sites in breast cancer, with more than 200 thousand new cases of invasive cancer diagnosed in the US alone in 2011. We set out to establish a multimodality imaging platform for bone metastases in small animals as a tool to non-invasively quantify metastasis growth, imaging the ensuing bone lesions and possibly the response to treatment. To this end, a mouse model of osteolytic metastatic bone tumors was characterized with SPECT/CT and MRI over time. A cell line capable of forming bone metastases, MDA-MB-231, was genetically modified to stably express the reporter gene herpes simplex virus-1 thymidine kinase (hsv-1 tk). The intracellular accumulation of the radiolabeled tracer [(123)I]FIAU promoted by HSV-1 TK specifically pinpoints the location of tumor cells which can be imaged in vivo by SPECT. First, a study using tumors implanted subcutaneously was performed. The SPECT/MRI overlays and the ex vivo γ-counting showed a linear correlation in terms of %ID/cm(3) (R(2)=0.93) and %ID/g (R(2)=0.77), respectively. Then, bone metastasis growth was imaged weekly by SPECT/CT and T2-weighted MRI over a maximum of 40 days post-intracardiac injection of tumor cells. The first activity spots detectable with SPECT, around day 20 post-cell injection, were smaller than 2mm(3) and not yet visible by MRI and increased in volume and in %ID over the weeks. Osteolytic bone lesions were visible by CT (in vivo) and μCT (ex vivo). The SPECT/MRI overlays also showed a linear correlation in terms of %ID/cm(3) (R(2)=0.86). In conclusion, a new multimodality imaging platform has been established that non-invasively combines images of active tumor areas (SPECT), tumor volume (MRI) and the corresponding bone lesions (CT and μCT). To our knowledge this is the first report where the combination of soft tissue information from MRI, bone lesions by CT, and reporter gene imaging by SPECT is used to non-invasively follow metastatic bone lesions.