Simian T-lymphotropic retroviruses with structural, antigenic, and cytopathic features similar to the etiologic agent of human acquired immunodeficiency syndrome, human T-lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV), have been isolated from a variety of primate species including African green monkeys (STLV-IIIAGM). This report describes nucleic acid cross-reactivity between STLV-IIIAGM and HTLV-III/LAV, molecular cloning of the STLV-IIIAGM genome, and evaluation of its structure and genetic relationship to other retroviruses. Overlapping clones from a cell line infected with virus from a single animal were found to encompass the entire STLV-IIIAGM genome and exhibit a limited degree of restriction-site variability. Specific hybridizing fragments were detected in DNA from this and other STLV-IIIAGM-infected cell lines. A fraction of viral DNA present in at least two STLV-IIIAGM lines persists as unintegrated viral DNA, a characteristic of infection with cytopathic retroviruses. Strongest cross-reactivity was detected between HTLV-III/LAV pol- and gag- genes and STLV-IIIAGM, whereas no cross-reactivity was detected between STLV-IIIAGM and molecular clones of human T-lymphotropic virus types I and II (HTLV-I and -II), visna virus, bovine leukemia virus, or feline leukemia virus.