The nucleocapsid (NC) protein from HIV-1 contains two zinc-fingers, both of which are necessary for virus replication. This is the first in-depth study that presents the effects of nucleocapsid zinc-finger substitutions on the kinetics of reverse transcription and integration. Over a 72-h time-course of infection, the quantities of viral DNA (vDNA) observed with viruses containing either the nucleocapsid His23Cys or His44Cys mutations were significantly lower than those observed in infections with virus containing wild-type NC. In addition, the kinetics of vDNA formation and loss were significantly different from wild-type. The kinetic profiles observed indicated reduced vDNA stability, as well as defects in reverse transcription and integration. Overall, the defect in integration was much more pronounced than the reverse transcription defects. This suggests that the principal reason for the replication defectiveness of these mutant viruses is impairment of integration, and thus demonstrates the critical importance of NC in HIV-1 infection.