The development of acquired immunodeficiency syndrome or of acquired immunodeficiency syndrome-related complex by transmission of human T-lymphotropic retrovirus III by semen has previously been implicated by epidemiological studies. In vitro investigations were performed on mononuclear cells obtained from the semen of patients with acquired immunodeficiency syndrome to identify human T-lymphotropic retrovirus III or related retrovirus. The presence of human T-lymphotropic retrovirus III was demonstrated (a) in primary cell cultures, by the detection of the Mr 24,000 protein by indirect immunofluorescence assays by Day 6; (b) in activated long-term cell culture by reverse transcriptase activity, by indirect immunofluorescence (Mr 24,000 protein); and (c) in cocultures of T-cells from semen of AIDS patients and H9 cells by reverse transcriptase activity, indirect immunofluorescence, and the presence of virus particles by electron microscopy.