The HIV-1 nucleocapsid protein (NC) has been hypothesized to be cleaved by the viral protease (PR) during early infection. Characterization of viruses, with amino-acid substitutions that modulate PR cleavage of NC in vitro, was performed in cell culture. Two of the NC mutants, NCN17F and NCN17G, had decreased infectivity and exhibited severe H9 replication defects. Examination of viral DNA after infections revealed defects in reverse transcription and integration, although integration defects were cell-type dependent. However, while the defects in reverse transcription and integration correlate with lowered infectivity in a single-round of infection, they did not approach the magnitude of the replication defect measured in H9 cells over multiple rounds. Importantly, we fail to see evidence that H9 cells are re-infected with the NCN17G and NCN17F viruses 24 h after the initial infection, which suggests that the principal defect caused by these NC mutations occurs during late events of viral replication.