During the assembly of human immunodeficiency virus type 1 (HIV-1) particles, the tetraspanin CD63 can be incorporated into the viral membrane. Indeed, cell surface tetraspanin microdomains, that include CD63, have been proposed as sites for virus release. In addition, antibodies against CD63 can inhibit HIV infection of macrophages. In this cell type, HIV assembles into intracellularly sequestered plasma membrane domains that contain several other tetraspanins including CD81, CD9 and CD53. CD63 is recruited to this domain following HIV infection. Together these observations suggest that CD63 may have some function in the assembly of infectious virus particles and/or the infectivity of assembled virions. Here we have used RNA interference to knock down CD63 expression in monocyte derived primary macrophages. We show that in the absence of CD63, HIV assembly is quantitatively comparable to that seen in CD63 expressing macrophages and that virus assembly occurs on compartments positive for CD81, CD9 and CD53. Moreover, the infectivity of macrophage-derived virus is unaffected by the loss of CD63. Together our results indicate that, at least in tissue culture, CD63 expression is not required for either the production or the infectivity of HIV-1.