Human immunodeficiency virus type 1 (HIV-1) assembly, budding, and release occur mostly at the plasma membrane in T lymphocytes as well as in established nonlymphoid cell lines, while in macrophages these processes occur primarily in intracellular compartments that harbor late endosomal/multivesicular body (LE/MVB) markers, including human leukocyte antigen DR (HLA-DR). Major histocompatibility complex class II molecules (MHC-II), which are expressed in macrophages and activated T cells, have been previously reported to induce the formation of multilaminar and multivesicular endocytic MHC-II-like structures analogous to MVB upon their expression in HEK 293 cells. Here, we have examined the role of MHC-II in HIV-1 Gag targeting as well as in virus assembly and release. Expression of HLA-DR in nonlymphoid cell lines induced a relocation of Gag to intracellular compartments that harbored LE/MVB markers and increased the accumulation of viral particles assembling intracellularly. Consequently, viral production and release from the cell surface was found to be substantially decreased in HLA-DR-expressing cells. This process was specific, since it was not observed with HLA-DR molecules lacking their cytoplasmic tails, nor with structurally related but functionally distinct MHC-II molecules such as HLA-DM or HLA-DO. Importantly, virus released intracellularly in HLA-DR-expressing cells retained infectivity. Overall, these results suggest a role of MHC-II molecules in promoting HIV-1 assembly and budding to LE/MVB and raise the possibility that this activity might be part of a normal pathway of virus production in cell types physiologically expressing MHC-II molecules, such as macrophages.