The nucleocapsid protein (NC) of retroviruses is involved in viral RNA packaging and initiation of reverse transcription. NC also mediates interactions between Gag and actin filaments. We found that residues at the amino terminus of NC are involved in efficient actin binding. When alanine residues were substituted for the arginine and lysine at positions 10 and 11 of NC in HIV(NL4-3), these mutations decreased actin binding but had only a modest effect on virus infectivity. A similarly mutated virus based on the HXB2 clone of HIV was not infectious. Mutational analysis of NL4-3 NC residues demonstrated that NC polymorphisms modulated the phenotype of NC mutations. Conservative amino acid differences between HXB2 and NL4-3 NCs were sufficient to explain the difference in infectivity of viruses carrying the R10A and K11A mutations.