The matrix domain (MA) of human immunodeficiency virus type 1 Pr55Gag is covalently modified with a myristoyl group that mediates efficient viral production. However, the role of myristoylation, particularly in the viral entry process, remains uninvestigated. This study replaced the myristoylation signal of MA with a well-studied phosphatidylinositol 4,5-biphosphate-binding plasma membrane (PM) targeting motif, the phospholipase C-delta1 pleckstrin homology (PH) domain. PH-Gag-Pol PM targeting and viral production efficiencies were improved compared with Gag-Pol, consistent with the estimated increases in Gag-PM affinity. Both virions were recovered in similar sucrose density-gradient fractions and had similar mature virion morphologies. Importantly, PH-Gag-Pol and Gag-Pol pseudovirions had almost identical infectivity, suggesting a dispensable role for myristoylation in the virus life cycle. PH-Gag-Pol might be useful in separating the myristoylation-dependent processes from the myristoylation-independent processes. This the first report demonstrating infectious pseudovirion production without myristoylated Pr55Gag.