Antisera to 21 synthetic peptides containing hydrophilic sequences of simian immunodeficiency virus strain mac251 (SIVmac251) gp120 and gp32 were tested for the ability to neutralize SIVmac251. Goat antisera raised to peptides SP-1 and SP-1V containing the carboxy-terminal portion of the V3 domain of SIVmac251 gp120 between amino acids 327 and 339 inhibited syncytium formation (90% inhibition at a 1/1024 dilution) and cell killing of CEMx174 cells by SIVmac251 (50%) inhibition of cell killing at a dilution of 1/5832), SIVDeltaB670 (1/568), and SIVsmH4 (1/740). Neutralizing antibodies to SIVmac251, SIVDeltaB670, and SIVsmH4 could be adsorbed by peptides containing a neutralizing V3 sequence of SIVmac251 gp120 (GLVFHSQPIND, amino acids 329-339) but not by peptides lacking this sequence. This V3 neutralizing region corresponds to a homologous V3 neutralizing site within HIV-2 gp120 reported by Björling et al. 1991, Proc. Natl. Acad. Sci. USA 88, 6082-6086, 1994, J. Immunol. 152, 1952-1959). Antibodies in 20 of 31 sera obtained from rhesus macaques infected with SIVmac251 reacted with a peptide containing the entire V3 sequence of SIVmac251 gp120, whereas no sera contained antibodies reacting with the V3 neutralizing site between amino acids 329 and 339. Low levels of antibody-mediated recognition and subsequent lack of selective pressure against this linear V3 neutralizing site might in part explain why this region is not a dominant neutralizing site and also why sequences within V3 do not vary during the course of SIV infection.