We recently reported the isolation and characterization of a novel human T-lymphotropic retrovirus, HTLV-III, in patients with acquired immune deficiency syndrome (AIDS) and in those at risk for the disease. After extensive sero-epidemiological studies, together with numerous virus isolations from these patients, we concluded that HTLV-III is the causative agent of AIDS. Here we report the molecular cloning and characterization of two highly related but distinct forms of the HTLV-III genome. The viral genome is approximately 10 kilobases long and is detected in HTLV-III-infected cells but not in uninfected cells, including normal human tissue, indicating that this virus is exogenous to man. We also demonstrate distant nucleic acid sequence homology between the cloned genome of HTLV-III and those of HTLV-I and HTLV-II. The availability of the cloned HTLV-III genome will now allow an unambiguous comparison of this virus with other retroviruses that also have been associated with the pathogenesis of AIDS, and moreover, with facilitate the development of diagnostic and therapeutic measures in the treatment of AIDS.