We have isolated a molecular clone of the full-length integrated provirus of simian acquired immune deficiency syndrome retrovirus serotype 1 (SRV-1) from a fatal case of simian acquired immune deficiency syndrome in a juvenile rhesus macaque. An integrated SRV-1 provirus was cloned, sequenced, and found to contain four large open reading frames encoding gag-precursor protein, protease, polymerase, and envelope. The proviral clone was transfected into D17 canine osteosarcoma cells and found to produce infectious virus. A comparison of the sequences of this clone with a noninfectious clone showed 20 differences, resulting in 10 amino acid changes. Also, a cluster of exchanges, short insertions, and deletions in the 5' leader sequences resulted in extension of the tRNA(Lys) primer-binding site from 14 to 19 nucleotides. Virus isolated from transfected cells was shown to be infectious and pathogenic, resulting in disease that followed the same time course and mortality as disease induced by uncloned, in vitro cultivated virus isolated from diseased animals. These results unequivocally show that a type D retrovirus (SRV-1) causes a fatal immunosuppressive syndrome in rhesus monkeys.