Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of adolescence and childhood. Because RMS tumors are highly vascularized, we sought to determine which factors secreted by RMS cells are crucial in stimulating angiogenesis in response to hypoxia. To address this issue, we evaluated expression of several proangiogenic factors [interleukin (IL)-8, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF)-2, stromal-derived factor (SDF)-1, hepatocyte growth factor (HGF) and leukemia inhibitory factor (LIF)] in 8 human RMS cell lines in both normal steady-state and hypoxic conditions. We found by real-time quantitative polymerase chain reaction (RQ-PCR) and confirmed by enzyme-linked immunosorbent assay (ELISA) that from all the factors evaluated, IL-8, whose expression is very low in normoxia, had been very highly expressed and secreted by RMS cells lines during hypoxic conditions ( approximately 40-170 times). Interestingly, this upregulation was not affected by knocking down hypoxia-inducible factor (HIF)-1alpha, but was inhibited by mitogen-activated protein kinase (MAPK)p42/44 and phosphatidylinositaol 3-kinase (PI3K)/AKT pathway inhibitors. This suggests that IL-8 expression is regulated in an activating protein (AP)-1- and nuclear factor (NF)-kappaB-dependent manner. Furthermore, we found that conditioned media (CM) harvested from RMS cells exposed to hypoxia activated and stimulated chemotactic responses in human umbilical vein endothelial cells (HUVECs) and that IL-8 was responsible for hypoxia-related effects. Finally, by employing shRNA, the expression of IL-8 in human RH-30 cells was downregulated. We noticed that such RMS cells, if injected into skeletal muscles of immunodeficient mice, have a reduced ability for tumor formation. We conclude that IL-8 is a pivotal proangiogenic factor released by human RMS cells in hypoxic conditions and that the targeting of IL-8 may prove to be a novel and efficient strategy for inhibiting RMS growth.