Cell-surface-associated proteolysis plays a crucial role in embryonic development, monocyte/macrophage recruitment and tumour cell invasion. The glycolytic enzyme ENO-1 (enolase-1) is translocated from the cytoplasm to the cell surface, where it binds PLG (plasminogen) to enhance pericellular plasmin production and cell motility. In the present study, ENO-1 was found to localize to a specialized subset of lipid rafts called caveolae as demonstrated by fluorescence confocal microscopy and sucrose gradient ultracentrifugation. Co-immunoprecipitation studies revealed that ENO-1 interacts with Cav-1 (caveolin-1), but not with Cav-2, via the CSD (Cav-scaffolding domain). Moreover, an evolutionarily conserved CBM (Cav-binding motif) F296DQDDWGAW304 was identified within ENO-1. The point mutation W301A within the ENO-1 CBM was, however, not sufficient to disrupt ENO-1-Cav-1 interaction, whereas the mutations F296A and W304A markedly affected ENO-1 protein expression. Furthermore, ENO-1 was found associated with Annx2 (annexin 2), representing another caveolar protein, and this interaction was dependent on Cav-1 expression. Knockdown of Cav-1 and Annx2 markedly decreased cell surface expression of ENO-1. ENO-1 overexpression increased cell migration and invasion in a Cav-1-dependent manner. Thus the differential association of ENO-1 with caveolar proteins regulates ENO-1 subcellular localization and, consequently, ENO-1-dependent cell migration and invasion.