The growth kinetics of wild-type and nef mutant viruses of human immunodeficiency virus type 1 were comparatively analysed in several human CD4(+) cell lines. Delayed replication of nef mutant virus was observed in all cell lines examined. To determine the stage in the virus replication cycle that is affected by Nef, a single-round replication assay was performed. Initially, the expression of marker genes in transfected cells was examined in order to study the role of Nef in the late phase of infection, The results obtained indicated that Nef is dispensable during the transcription to virion production stage. Next, the effect of Nef on the early phase was investigated with a single-round infection. It was demonstrated that Nef is required in the early phase of the virus replication cycle, from virion adsorption to integration. Finally, the infectivity of virus stocks prepared from four cell lines was determined, The relative infectivity of the nef mutant from the four cell lines differed, Taken together, we conclude that Nef acts via modulation of viral particles to enhance virus infectivity in a cell-dependent manner.