During the last 2 yr more than 100 independent isolates of human T-cell leukemia virus type III have been obtained in this laboratory. Most isolates were from peripheral blood T-lymphocytes established in cell culture from acquired immunodeficiency syndrome (AIDS) and acquired immunodeficiency syndrome-related complex (ARC) patients and healthy donors at risk for AIDS. Several were also obtained from leukocytes from bone marrow, lymph node, and brain tissue and from body excretions, e.g., saliva and semen. In addition HTLV-III was found in cell-free plasma. The incidence (number of isolates per number of patients or donors tested) of virus isolation was approximately 80% for ARC patients, approximately 50% for AIDS patients, and approximately 30% for healthy individuals at risk for AIDS. Inclusion of hydrocortisone in cell culture medium greatly facilitated the isolation of virus from primary leukocytes from AIDS/ARC patients and also promoted its transmission to fresh leukocytes in vitro. Biological analysis of cells from infected patients or donors, as well as from normal peripheral blood mononuclear cells exposed to virus in vitro, demonstrated that OKT4/leu3a+ T-lymphocytes were preferentially infected and were subjected to a characteristic cytopathic effect. In addition to the well-defined individuals at risk for AIDS, heterosexual transmission of HTLV-III with its subsequent pathological manifestations was found. Virus was isolated from males with heterosexual promiscuity as their only recognized risk factor and from the spouses of these and other AIDS and ARC patients.