The N-terminally myristoylated matrix (MA) domain of the HIV-1 Gag polyprotein promotes virus assembly by targeting Gag to the inner leaflet of the plasma membrane. Recent studies indicate that, prior to membrane binding, MA associates with cytoplasmic tRNAs (including tRNA), and in vitro studies of tRNA-dependent MA interactions with model membranes have led to proposals that competitive tRNA interactions contribute to membrane discrimination. We have characterized interactions between native, mutant, and unmyristylated (myr-) MA proteins and recombinant tRNA by NMR spectroscopy and isothermal titration calorimetry. NMR experiments confirm that tRNA interacts with a patch of basic residues that are also important for binding to the plasma membrane marker, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P]. Unexpectedly, the affinity of MA for tRNA (K = 0.63 ± 0.03 μM) is approximately 1 order of magnitude greater than its affinity for PI(4,5)P-enriched liposomes (K = 10.2 ± 2.1 μM), and NMR studies indicate that tRNA binding blocks MA association with liposomes, including those enriched with PI(4,5)P, phosphatidylserine, and cholesterol. However, the affinity of MA for tRNA is diminished by mutations or sample conditions that promote myristate exposure. Since Gag-Gag interactions are known to promote myristate exposure, our findings support virus assembly models in which membrane targeting and genome binding are mechanistically coupled.