We examined the association of HTLV-1 Gag with tetraspanin-enriched microdomains in the plasma membrane. Immunofluorescent staining and confocal image analysis showed that HTLV-1 Gag protein colocalized with CD82 and other tetraspanins at the plasma membrane of T cells. HTLV-1 Gag, which is associated with the inner surface of the plasma membrane, was concentrated to the patches formed by antibody-mediated cross-linking of CD82 on the cell surface. Also, CD82 and HTLV-1 Gag rapidly segregated to the immune synapse that is formed between Raji B cells and Jurkat T cells in the presence of bacterial superantigen. CD82, which was immunoprecipitated from cell extracts prepared in Brij97 detergent conditions, was associated with the matrix (MA) protein. Stable interaction of MA and CD82 in Brij97-disrupted cell extracts required Gag multimerization and proteolytic processing. The form of MA that coimmunoprecipitated with CD82 was a cysteine-linked homodimer. The viral envelope glycoprotein was not required for the association of Gag with CD82-enriched membrane regions. In contrast to HTLV-1, HIV-1 Gag did not colocalize, cosegregate, or coimmunoprecipitate with CD82. Our data suggest that once at the plasma membrane, HTLV-1 virion components associate with CD82-containing microdomains, which may facilitate the mobilization of nascent virions to sites of intercellular adhesion.