We have previously shown that the chemokine fractalkine promotes the adhesion of human prostate cancer cells to bone marrow endothelial cells as well as their migration toward human osteoblasts in vitro. Thus, the interaction of fractalkine with its receptor CX3CR1 could play a crucial role in vivo by directing circulating prostate cancer cells to the bone. We found that although CX3CR1 is minimally detectable in epithelial cells of normal prostate glands, it is overexpressed upon malignant transformation. Interestingly, osteoblasts, stromal and mesenchymal cells derived from human bone marrow aspirates express the cell-bound form of fractalkine, whereas the soluble form of the chemokine is detected in bone marrow supernatants. To investigate the mechanisms regulating the levels of soluble fractalkine in the bone marrow, we focused on androgens, which play a critical role in both prostate cancer progression and skeletal metastasis. Here, we show that dihydrotestosterone dramatically increases the cleavage of fractalkine from the plasma membrane of bone cells and its action is reversed by nilutamide—an antagonist of the androgen receptor—as well as the wide-spectrum inhibitor of matrix metalloproteases, GM6001. However, dihydrotestosterone was unable to induce fractalkine-cleavage from human bone marrow endothelial cells. Thus, androgens could promote the extravasation of CX3CR1-bearing cancer cells on a fractalkine concentration gradient, while leaving unaltered their ability to adhere to the bone marrow endothelium. In conclusion, our results indicate that CX3CR1, fractalkine, and the enzymes responsible for its cleavage might represent suitable targets for therapies aiming to counteract skeletal secondary tumors from prostate adenocarcinoma. [Cancer Res 2008;68(6):1715–22]