The skeleton is a preferred site for breast cancer metastasis. We have developed a multimodality imaging approach to monitor the transforming growth factor β (TGFβ) signaling pathway in bone metastases, sequentially over time in the same animal. As model systems, two MDA-MB-231 breast cancer cells lines with different metastatic tropisms, SCP2 and SCP3, were transduced with constitutive and TGFβ-inducible reporter genes and were tested in vitro and in living animals. The sites and expansion of metastases were visualized by bioluminescence imaging using a constitutive firefly luciferase reporter, while TGFβ signaling in metastases was monitored by microPET imaging of HSV1-TK/GFP expression with [18F]FEAU and by a more sensitive and cost-effective bioluminescence reporter, based on nonsecreted Gaussia luciferase. Concurrent and sequential imaging of metastases in the same animals provided insight into the location and progression of metastases, and the timing and course of TGFβ signaling. The anticipated and newly observed differences in the imaging of tumors from two related cell lines have demonstrated that TGFβ signal transduction pathway activity can be noninvasively imaged with high sensitivity and reproducibility, thereby providing the opportunity for an assessment of novel treatments that target TGFβ signaling.—Serganova, I., Moroz, E., Vider, J., Gogiberidze, G., Moroz, M., Pillarsetty, N., Doubrovin, M., Minn, A., Thaler, H. T., Massague, J., Gelovani, J., Blasberg, R. Multimodality imaging of TGFβ signaling in breast cancer metastases.