The chemokine receptor CXCR4 is functionally expressed on the cell surface of various cancer cells, and plays a role in cell proliferation and migration of these cells. Specifically, in breast cancer cells the CXCR4/CXCL12 axis has been implicated in cell migration in vitro and in metastasis in vivo, but the underlying signaling mechanisms are incompletely understood. The xenograft-derived MDA-MB-231 breast cancer cell line (231mfp), which was shown previously to grow more aggressively than the parent cells, showed increased CXCR4 expression at the mRNA, total protein and cell surface expression level. This correlated with an enhanced response to CXCL12, specifically in augmented and prolonged Akt activation in a G(i), Src family kinase and PI-3 kinase dependent fashion. 231mfp cells migrated towards CXCL12--in contrast to the parent cell line--and this chemotaxis was blocked by inhibition of G(i), Src family kinases, PI-3 kinase and interestingly, Akt itself, as could be shown with two pharmacological inhibitors, a dominant negative Akt construct and with Akt shRNA. Collectively, we have demonstrated that prolonged Akt activation is an important signaling pathway for breast cancer cells expressing CXCR4 and is necessary for CXCL12-dependent cell migration.