The genome of the human immunodeficiency virus HIV-1 contains at least eight genes, of which three (sor, R, and 3′orf) have no known function. In this study, the role of the sor gene was examined by constructing a series of proviral genomes of HIV-1 that either lacked the coding sequences for sor or contained point mutations in sor. Analysis of four such mutants revealed that although each done could generate morphologically normal virus particles upon transfection, the mutant viruses were limited in their capacity to establish stable infection. Virus derived from transfection of Cos-1 cells (OKT4 -) with sor mutant proviral DNA's was resistant to transmission to OKT4+ "susceptible" cells under cell-free conditions, and was transmitted poorly by coculture. In contrast, virus derived from clones with an intact sor frame was readily propagated by either approach. Normal amounts of gag-, env-, and pol-derived proteins were produced by all four mutants and assays in both lymphoid and nonlymphoid cells indicated that their trans-activating capacity was intact and comparable with wild type. Thus the sor gene, although not absolutely required in HIV virion formation, influences virus transmission in vitro and is crucial in the efficient generation of infectious virus. The data also suggest that sor influences virus replication at a novel, post-translational stage and that its action is independent of the regulatory genes tat and trs.