We screened monoclonal antibodies to cell-surface proteins and selected an antibody, called 6H1, that recognizes a putative integrin-associated protein. The 6H1 monoclonal antibody (mAb) indirectly coprecipitated alpha 3 beta 1 and/or alpha 6 beta 1, but not alpha 2 beta 1, or alpha 5 beta 1 from Brij 96 detergent lysates of multiple cell lines. Large scale purification using the 6H1 mAb yielded a single protein of 45-60 kDa with an amino-terminal sequence that exactly matched CD63. Confirming that the 6H1 mAb recognized the CD63 protein, 6H1 and a known anti-CD63 mAb yielded identical coprecipitation results and identical colocalization into lysosomal granules containing cathepsin D. Furthermore, we used an established anti-CD63 mAb to detect this protein in an alpha 3 beta 1 immunoprecipitate, and also we observed VLA-3 and CD63 colocalization in cellular "footprints." Notably, the cytoplasmic domain of alpha 3 was neither required nor sufficient for CD63 association, suggesting that it occurred elsewhere within the alpha 3 beta 1 complex. Knowledge of these specific CD63-alpha 3 beta 1 and CD63-alpha 6 beta 1 biochemical associations should lead to critical insights into the specialized functions of alpha 3 beta 1, alpha 6 beta 1, and CD63.