We hypothesized that the CXC chemokine receptor-4 (CXCR4)–stromal-derived factor-1 (SDF-1) axis may be involved in metastasis of CXCR4⁺ tumor cells into the bone marrow and lymph nodes, which secrete the α-chemokine SDF-1. To explore this hypothesis, we phenotyped by fluorescence-activated cell sorter analysis various human tumor cell lines for expression of CXCR4 and found that it was highly expressed on several rhabdomyosarcoma (RMS) cell lines. We also observed that cell lines derived from alveolar RMS, which is characterized by recurrent PAX3- andPAX7-FKHR gene fusions and is associated with a poor prognosis, expressed higher levels of CXCR4 than lines derived from embryonal RMS. Furthermore, transfer of a PAX3-FKHRgene into embryonal RMS cell activates CXCR4 expression. Because alveolar RMS frequently metastasizes to the bone marrow and lymph nodes, it seems that the CXCR4–SDF-1 axis could play an important role in this process. These findings prompted us to determine whether SDF-1 regulates the metastatic behavior of RMS cells. Accordingly, we found that, although SDF-1 did not affect proliferation or survival of these cell lines, it induced in several of them (1) phosphorylation of mitogen-activated protein kinase p42/44; (2) locomotion; (3) directional chemotaxis across membranes covered by laminin, fibronectin, or Matrigel; (4) adhesion to laminin, fibronectin, and endothelial cells; and (5) increased MMP-2 and diminished tissue inhibitors of metalloproteinases secretion. The small-molecule CXCR4-specific inhibitor, T140, effectively blocked the in vitro responses of RMS cells to SDF-1. On the basis of these observations we suggest that the CXCR4–SDF-1 axis may play an important role in tumor spread and metastasis of RMS cells to bone marrow and that molecular strategies aimed at inhibiting this axis could thus prove to be useful therapeutic measures.