In the replication of human immunodeficiency virus type 1 (HIV-1), gag MA (matrix), a major structural protein of the virus, carries out opposing targeting functions. During virus assembly, gag MA is cotranslationally myristoylated, a modification required for membrane targeting of gag polyproteins. During virus infection, however, gag MA, by virtue of a nuclear targeting signal at its N terminus, facilitates the nuclear localization of viral DNA and establishment of the provirus. We now show that phosphorylation of gag MA on tyrosine and serine prior to and during virus infection facilitates its dissociation from the membrane, thus allowing it to translocate to the nucleus. Inhibition of gag MA phosphorylation either on tyrosine or on serine prevents gag MA-mediated nuclear targeting of viral nucleic acids and impairs virus infectivity. The requirement for gag MA phosphorylation in virus infection is underscored by our finding that a serine/threonine kinase is associated with virions of HIV-1. These results reveal a novel level of regulation of primate lentivirus infectivity.