Antigenic cross-reactivity of human T-cell leukemia virus type III (HTLV-III) with HTLV-I and HTLV-II and other retroviruses was measured by using a stringent homologous competition radioimmunoassay for the Gag protein p24 and a less stringent electrophoretic transfer blot assay. In the competition radioimmunoassay only minimal crossreactivities were detected between HTLV-III p24 and both HTLV-I and HTLV-II. No cross-reactivity was detected with any other retrovirus. In the electrophoretic transfer blot system using rabbit antibody to HTLV-I, HTLV-II, and HTLV-III, low-level cross-reaction was detected between HTLV-I and HTLV-IH and between HTLV-II and HTLV-IIl. Unlike the cross-reactivity between HTLV-I p24 and HTLV-III p24, which was bidirectional, the one between HTLV-II and HTLV-III was only a one-way reactivity. Antiserum to HTLV-II recognized HTLV-III p24, but the antiserum to HTLV-III did not recognize HTLV-II p24. The results indicate that HTLV-III is a unique retrovirus with a limited homology with HTLV-I and HTLV-II but unrelated to most other retroviruses.The human retrovirus human T-cell leukemia virus type III (HTLV-III) was isolated from cultured T cells of several patients with acquired immunodeficiency syndrome (AIDS), or generalized lymphadenopathy, and from asymptomatic carriers of the virus (1-3). Like HTLV-I and HTLV-II, this new isolate has a preferential tropism for OKT4+ lymphocytes and contains a Mg2+-dependent reverse transcriptase (1, 2) and a major core protein of about 24,000 daltons (4). Unlike the first two subgroups, however, HTLV-Il does not transform T cells in vitro and the in vitro transmission of the virus to fresh human T-cells only results in a transient virus production (1, 2, 5, 6). The virus could be conveniently propagated in an immortalized human neoplastic cell line (HT) and several clones of this cell line (1). Seroepidemiological assays using this virus showed a nearly complete correlation between AIDS and antibodies to HTLV-III antigens (3, 4, 7). In addition, nearly 90% of patients with lymphadenopathy syndrome and a significant but lower proportion ("40%) of asymptomatic homosexual men at risk of AIDS also carry circulating antibodies to HTLV-Ill. All of a group of patients with no risk factors for AIDS who developed AIDS after receiving blood transfusions from blood donors who were themselves seropositive showed HTLV-III antibodies (unpublished results). All of these data indicate HTLV-III to be the etiological agent for AIDS. A virus with similar properties, but described variously as lymphadenopathy-associated virus (9, 10), immune deficiency-associated virus I, and immune deficiency-associated virus II (11), has been isolated from patients with lymphadenopathy and AIDS. In the present communication we describe the antigenic characteristics of the major core protein of HTLV-III. We show that HTLV-III is a unique retrovirus with some, albeit limited, cross-reactivities with HTLV-I and HTLV-II, but not detectably related to other viruses. In addition, ...