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, alpha and beta, 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 HSP90alpha 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 HSP90alpha and -beta. Cell migration from cerebellar and sciatic nerve explants is inhibited by anti-HSP90alpha and anti-HSP90beta 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 alpha and beta isoforms of HSP90 reveals that HSP90 activity is associated with actin cytoskeletal organization, necessary for lamellipodia formation.