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 cross-reactivities 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-III and between HTLV-II and HTLV-III. 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.