Type 1 human immunodeficiency viruses encoding mutated nef reading frames are 10- to 30-fold less infectious than are isogenic viruses in which the nef gene is intact. This defect in infectivity causes nef-negative viruses to grow at an attenuated rate in vitro. To investigate the mechanism of Nef-mediated enhancement of viral growth rate and infectivity, a complementation analysis of nef mutant viruses was performed. To provide Nef in trans upon viral infection, a CEM derivative cell line (designated CLN) that expresses Nef under the control of the viral long terminal repeat was constructed. When nef-negative virus was grown in CLN cells, its growth rate was restored to wild-type levels. However, the output of nef-negative virus during the first 72 h after infection of CLN cells was not restored, suggesting that provision of Nef within the newly infected cell does not enhance the productivity of a nef-negative provirus. The genetically nef-negative virions produced by the CLN cells, however, were restored to wild-type levels of infectivity as measured in a syncytium formation assay in which CD4-expressing HeLa cells were targets. These trans-complemented, genetically nef-negative virions yielded wild-type levels of viral output following a single cycle of replication in primary CD4 T cells as well as in parental CEM cells. To define the determinants for producer cell modification of virions by Nef, the role of myristoylation was investigated. Virus that encodes a myristoylation-negative nef was as impaired in infectivity as was virus encoding a deleted nef gene. Because myristoylation is required for both membrane association of Nef and optimal viral infectivity, the possibility that Nef protein is included in the virion was investigated. Wild-type virions were purified by filtration and exclusion chromatography. A Western blot (immunoblot) of the eluate fractions revealed a correlation between peak Nef signal and peak levels of p24 antigen. Although virion-associated Nef was detected in part as the 27-kDa full-length protein, the majority of immunoreactive protein was detected as a 20-kDa isoform. nef-negative virus lacked both 27- and 20-kDa immunoreactive species. Production of wild-type virions in the presence of a specific inhibitor of the human immunodeficiency virus type 1 protease resulted in virions which contained only 27-kDa full-length Nef protein. These data indicate that Nef is a virion protein which is processed by the viral protease into a 20-kDa isoform within the virion particle.