We analyzed herein whether members of the tetraspanin superfamily are involved in human immature dendritic cell (DC) functions such as foreign antigen internalization, phagocytosis, and cell migration. We show that CD63, CD9, CD81, CD82, and CD151 are present in immature DCs. Whereas CD9 and CD81 are mostly expressed at the cell surface, CD63 and CD82 are also located in intracellular organelles. Complexes of monoclonal antibody (Mab) FC-5.01-CD63 or Fab-5.01-CD63 were rapidly translocated "outside-in" and followed the endocytic pathway through early endosomes and lysosomes, reaching major histocompatibility complex (MHC) class II-enriched compartments (MIICs) in less than one hour. Internalization of CD63 was also observed during Saccharomyces cerevisiae phagocytosis. Moreover, an association of CD63 with the beta-glycan receptor dectin-1 was observed. Mabs against CD9, CD63, CD81, and CD82 enhanced by 50% the migration induced by the chemokines macrophage inflammatory protein-5 (MIP-5) and MIP-1alpha. Concomitantly, Mabs against CD63 and CD82 diminished the surface expression of CD29, CD11b, CD18, and alpha5 integrins. By immunoprecipitation experiments we found that CD63 associated with integrins CD11b and CD18. These results suggest that CD9, CD63, CD81, and CD82 could play a role in modulating the interactions between immature DCs and their environment, slowing their migratory ability. However, only CD63 would intervene in the internalization of complex antigens.