Nef gene function is diverse among virus isolates of primate immunodeficiency viruses. We found differential effects of nef mutation on the virus replication between two HIV-1 clones, NL432 and LAI. The nef mutation in NL432 affected the infectivity more severely compared with that in LAI, although the Nef functions of both clones were comparable. Analysis with a series of chimeric viruses between NL432 and LAI revealed that the gag-pol region was responsible for the differential effect of nef mutation. The functional association between Nef and gag-pol suggested that one of the potential targets of Nef was located within the gag-pol region.