The major homology region (MHR) is a highly conserved sequence in the gag gene of all retroviruses, including HIV-1. Its role in assembly is unknown, but deletion of the motif significantly impairs membrane binding and viral particle formation. To begin characterizing this defect, we have determined the contribution of this region to the energetics of the assembly process. Intrinsic fluorescence studies were conducted to determine the change in free energy associated with membrane and RNA binding using tRNA and large unilamellar vesicles of 1-palmitoyl-2-oleoylphosphatidylserine as models. For the wild-type protein, the change in free energy was within RT [600 cal/(mol·K)] whether Gag binds first to RNA or to the membrane. Thus, the initial binding of Gag can be to either substrate, but in vivo conditions favor initial association to RNA presumably due to its higher local concentration. After establishing the pattern of assembly, we compared the binding energy of Gag_WT versus the deletion mutant, Gag_ΔMHR. Gag_WT bound to membranes with a 2-fold higher affinity than Gag_ΔMHR, and the binding to RNA was similar for the two proteins. Gag prebound to RNA or to membrane exhibited ∼2−4-fold greater binding affinity than Gag_ΔMHR for binding the membrane or RNA, respectively. Most importantly, the mutant was significantly impaired in its ability to self-associate on RNA or on membrane surfaces. This key role of the MHR in promoting productive protein−protein interactions was also seen in altered amounts of cleavage products and the lack of membrane-bound, RNA-containing replication intermediates in infected cells. These results suggest that Gag first binds to RNA and then assembles into a multimeric complex with a large membrane-binding face that facilitates subsequent membrane binding. Deletion of the MHR disrupts the protein−protein interactions required to complete this process.