We have previously shown that there were no gross deletions or obvious sequence abnormalities within nef of human immunodeficiency virus type 1 (HIV-1) in the 10 long-term survivors studied (Y. Huang, L. Zhang, and D. D. Ho, J. Virol. 69:93-100, 1995). Here we extend our study to examine these nef alleles in a functional context. Using a new technique, termed site-directed gene replacement, we have precisely replaced the nef of an infectious molecular clone, HIV-1HXB2, with nef alleles derived from 10 long-term survivors as well as from a patient with AIDS. The replication properties of these chimeric viruses demonstrated that the nef alleles derived from long-term survivors neither significantly increased nor decreased viral replication, compared with the nef allele of Nef+ HIV-1HXB2 and that derived from a patient with AIDS. However, Nef+ viruses always replicated faster than virus lacking nef. Moreover, single-cell infection analysis by the MAGI assay showed that these chimeric viruses, as well as Nef+ HIV-1HXB2, were more infectious than Nef- HIV-1HXB2 was. Therefore, we conclude that the genotypic and phenotypic features of nef are not likely to account for the nonprogression of HIV-1 infection in the 10 cases studied, unless the function of the nef gene in vivo is not accurately reflected by the in vitro assays we used.