Heat shock protein HSP90 plays important roles in cellular regulation, primarily as a chaperone for a number of key intracellular proteins. We report here that the two HSP90 isoforms, ␣ and , also localize on the surface of cells in the nervous system and are involved in their migration. A 94-kDa surface antigen, the 4C5 antigen, which was previously shown to be involved in migration processes during development of the nervous system, is shown to be identical to HSP90␣ using mass spectrometry analysis. This identity is further confirmed by immunoprecipitation experiments and by induction of 4C5 antigen expression in heat shock-treated embryonic rat brain cultures. Moreover, immunocytochemistry on live cerebellar rat cells reveals cell surface localization of both HSP90␣ and -. Cell migration from cerebellar and sciatic nerve explants is inhibited by anti-HSP90␣ and anti-HSP90 antibodies, similarly to the inhibition observed with monoclonal antibody 4C5. Moreover, immunostaining with rhodamine-phalloidin of migrating Schwann cells cultured in the presence of antibodies against both ␣ and  isoforms of HSP90 reveals that HSP90 activity is associated with actin cytoskeletal organization, necessary for lamellipodia formation.The development of the vertebrate nervous system depends on extensive cell migration, which allows different cell types to reach their final destination and establish the composite organization of the adult nervous system. Cell migration is a complex process, which requires the coordination of many molecular and cellular events such as cell-cell recognition, adhesion, transmembrane signaling, and cell motility (1-23). The mechanisms underlying these processes involve orchestrated interactions between a large number of molecules (10, 24). Emerging evidence suggests that many proteins in the nervous system have multiple distinct functions. Thus, molecules involved in migration processes have been shown to participate additionally in other developmental events. These include the cell adhesion molecules (CAMs), which regulate axonal growth and regeneration (25); integrins, which mediate cell proliferation and synaptic plasticity (26); and neuregulins that promote neuronal differentiation and regulate glial commitment, proliferation, survival, and differentiation (27). We report here that heat shock protein 90 (HSP90) 1 is also a multifunctional protein, processing a novel role in neuronal migration. HSP90 is a highly conserved molecule with a wide distribution in various species. It acts as capacitor of morphological evolution in Drosophila melanogaster (28 -30). It also functions as a chaperone in unstressed cells, specifically involved in the folding or conformational regulation of central signal transduction molecules, including steroid hormone receptors and proto-oncogene kinases (31, 32). Moreover, it is a protective agent under stress conditions and is involved in protein renaturation and refolding (33). In the developing mammalian nervous system, HSP90 is highly expressed in the G 0 phase ...