The matrix domain (MA) is important for targeting of human immunodeficiency virus type 1 Gag assembly to the plasma membrane, envelope incorporation into virions, preintegration complex import into the nucleus, and nuclear export of viral RNA. Myristylation and phosphorylation are key regulatory events for MA function. Previous studies have indicated that MA phosphorylation at serine (Ser) residues is important for viral replication. This study defines the molecular mechanisms of virus particle assembly and infectivity through a detailed study of the role of MA serine phosphorylation. We show that the combined mutation of Ser residues at positions 9, 67, 72, and 77 impairs viral infectivity in dividing and nondividing cells, although the assembly of these Ser mutant viruses is comparable to that of wild-type virus. This defect can be rescued by pseudotyping these mutant viruses with vesicular stomatitis virus G protein, suggesting that these serine residues are critical in an early postentry step of viral infection. The phosphorylation level of MA in defective mutant viruses was severely reduced compared to that of the wild type, suggesting that phosphorylation of Ser-9, -67, -72, and -77 is important for an early postentry step during virus infection.