An extensive investigation into the initial association of HIV-1 Gag with lipid membranes was conducted with second harmonic generation. The roles of the lipid phase, phospholipid 1,2-dioleoylsn-glycero-3-phospho-(1-myo-inositol-4,5-bisphosphate) [PI(4,5)P-2], the presence of the myristoyl group on Gag, the C-terminus of Gag, and the presence of transfer ribonucleic acid (tRNA) in Gag-membrane association were examined using the physiologically most relevant full-length Gag protein studied thus far. The tighter packing of a bilayer composed of gel-phase lipids was found to have a lower relative amount of membrane-bound Gag in comparison to its fluid-phase counterpart. Rather than driving membrane association of Gag, the presence of PI(4,5)P-2 and the myristoyl group were found to anchor Gag at the membrane by decreasing the rate of desorption. Specifically, the interaction with PI(4,5)P(2 )allows Gag to overcome electrostatic repulsion with negatively charged lipids at the membrane surface. This behavior was verified by measuring the binding properties of Gag mutants in the matrix domain of Gag, which prevented anchoring to the membrane either by blocking interaction with PI(4,5)P-2 or by preventing exposure of the myristoyl group. The presence of tRNA was found to inhibit Gag association with the membrane by specifically blocking the PI(4,5)P-2 binding region, thereby preventing exposure of the myristoyl group and precluding subsequent anchoring of Gag to the membrane. While Gag likely samples all membranes, only the anchoring provided by the myristoyl group and PI(4,5)P-2 allows Gag to accumulate at the membrane. These quantitative results on the kinetics and thermodynamics of Gag association with lipid membranes provide important new information about the mechanism of Gag-membrane association.