The role of the cytoplasmic domain of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins in virus replication was investigated. Deletion of residues 840 to 856 at the carboxyl terminus of gp41 reduced the efficiency of virus entry during an early step in the virus life cycle between CD4 binding and formation of the DNA provirus without affecting envelope glycoprotein synthesis, processing, or syncytium-forming ability. Deletion of residues amino terminal to residue 846 was associated with decreased stability of envelope glycoproteins made in COS-1 cells, but this phenotype was cell type dependent. The cytoplasmic domain of gp41 was not required for the incorporation of the HIV-1 envelope glycoproteins into virions. These results suggest that the carboxyl terminus of the gp41 cytoplasmic domain plays a role in HIV-1 entry other than receptor binding or membrane fusion. The cytoplasmic domain of gp41 also affects the stability of the envelope glycoprotein in some cell types.