Cancer metastasis suppressor KAI1/CD82 belongs to the tetraspanin superfamily and inversely correlates with the metastatic potential of a variety of cancers. The mechanism of KAI1/CD82-mediated metastasis suppression remains unclear. In this study, we found a M(r) 68,00 cell-surface protein physically associated with KAI1/CD82 and named it KASP: a KAI1/CD82-associated surface protein. Distinctive from known KAI1/CD82 associations that usually occur in the context of "tetraspanin web," the KAI1/CD82-KASP association is likely to be direct because it is: (a) highly stoichiometric; (b) stabilized by chemical cross-linking; and (c) independent of cholesterol-enriched lipid rafts. Therefore, KASP is one of the major transmembrane proteins that associates with KAI1/CD82. Consistent with the wide distribution of KAI1/CD82, KASP is expressed ubiquitously in human tissues. Through peptide sequencing, KASP was identified as an immunoglobulin superfamily member called EWI2 or PGRL. Although EWI2/PGRL has been found to associate with tetraspanins CD9 and CD81, it forms distinct complexes with different tetraspanins, and its association with KAI1/CD82 could be independent of CD81 and CD9. Overexpression of EWI2/PGRL in Du145 metastatic prostate cancer cells inhibits cell migration on both fibronectin- and laminin-coated substratum, indicating that EWI2/PGRL directly regulates cell migration. Furthermore, EWI2/PGRL synergizes KAI1/CD82 in inhibiting cell migration, indicating that EWI2/PGRL is likely required for the function of KAI1/CD82. In summary, we identified a major KAI1/CD82-associated protein, EWI2/PGRL, that is important for KAI1/CD82-mediated suppression of cancer cell migration.