By analogy to other retroviruses, the major envelope glycoprotein - gp120 - of HTLV-III/LAV is a probable target for neutralizing antibody. This antigen has been purified from H9 cells chronically infected with the HTLV-IIIB prototype strain. Several goats immunized with the gp120 produced antibodies that neutralized infection of H9 by the homologous virus isolate. These same sera failed to neutralize the divergent HTLV-IIIRF isolate. Individuals infected with HTLV-III/LAV commonly develop antibodies to gp120 which could be isolated using the gp120 antigen coupled to an immunoadsorbent resin. The antibody fraction that bound tightly to such a resin was found to neutralize the IIIB but not the RF isolate in a fashion similar to that of the goat anti-gp120 sera. However, the nonbinding fraction (effluent) from the resin also contained neutralizing activity which was able to block infection by both virus isolates with similar efficacy. Human antibodies to the other virus envelope gene product, the transmembrane gp41, were also affinity-purified utilizing the recombinant peptide 121, but they failed to influence infection by either virus isolate.