The incorporation of the envelope (env) glycoprotein of the human immunodeficiency virus type 1 (HIV-1) into budding virions has been proposed to be mediated by an interaction between its cytoplasmic domain and the matrix protein of HIV-1. However, this interaction was never directly demonstrated and its role in the biogenesis of HIV-1 virions is still debated. Here, a direct interaction is reported between the matrix protein of HIV-1 and the cytoplasmic domain of the env protein of HIV-1. No interaction was seen with the env cytoplasmic domain of other retroviruses. The region of the HIV-1 env involved in the interaction was delineated by mutagenesis and is comprised of the C-terminal 67 amino acid residues of env. These results, as well as the analysis of mutants of the matrix protein, suggest that the interaction between the HIV-1 env and matrix proteins accounts for the specific incorporation of the env glycoprotein into HIV-1 virions.