A cell clone (Hut-78/F12) chronically infected with a non-producer human immunodeficiency virus type 1 (HIV-1) variant showed an abnormal pattern of virus structural proteins and released no detectable virus particles. Exchanges of homologous parts of the F12/HIV provirus and a replication-competent HIV (strain NL4-3) were undertaken to define the genetic determinants of the F12/HIV phenotype. The non-infectious phenotype was reproduced by replacing an NL4-3 genomic fragment encoding the C terminus of gp 120 and the N terminus of gp41 with the corresponding parts of the F12/HIV provirus. Conversely, a much more extended genomic fragment (encompassing the vif, pol and env genes) was necessary to convert the F12/HIV phenotype. These results demonstrate that the F12/HIV non-producer phenotype is the result of mutations scattered along most of the genome, rendering the conversion to an infectious phenotype a very unlikely event. The F12/HIV genome is thus a reliable model for preclinical studies of anti-HIV gene therapy.