Three mouse monoclonal antibodies (mAb) were produced against soluble recombinant vaccinia virus gp140 from SIV-mac251. Two mAbs (1B9 and 6C11) were mapped at the aa 411-430 sequence within the V4 domain, and the third mAb (3C8) recognizes a conformation-dependent epitope on the external envelope glycoprotein. This was shown by its loss of reactivity in Western blot and ELISA with dithiothreitol-reduced gp140. mAb 3C8, but not 1B9 and 6C11, cross-reacts well with gp140 and gp125 from HIV-2ROD, indicating that this discontinuous epitope includes conserved regions localized within the external envelope glycoprotein. Analysis of the neutralizing activities of the mAbs showed that only mAb 1B9 is able to inhibit both syncytium formation and SIVmac251 infection of human peripheral blood lymphocytes.