One of the enduring problems in HIV-1 research is the mechanism of incorporation of the viral envelope (Env) glycoprotein into viral particles. Several models have been proposed ranging from an entirely passive process to a requirement for binding of Env by the matrix (MA) domain of the Gag precursor polyprotein. It is clear that specific regions within MA and Env play important roles, as mutations in these domains can prevent Env incorporation. We have identified a point mutation in MA that rescues a broad range of Env-incorporation defective mutations, located both in MA and in Env. Our investigations into the mechanism of rescue have revealed the importance of interactions between MA monomers at a trimeric interface. Our results are consistent with previously published crystallographic models and now provide functional support for the existence of MA trimers in the immature Gag lattice. Furthermore, as the modification of trimer interactions plays a role in the rescue of Env incorporation, we propose that MA trimerization and the organization of the MA lattice may be critical factors in Env incorporation.