Ten new monoclonal antibodies (MAbs) to SIV envelope were produced and characterized. Using a panel of 28 MAbs, 10 antibody binding sites on SIV envelope protein were identified. Seven sites were located in gp120 and three in gp41. Five sites in gp120 and two in gp41 were defined by overlapping peptides. The remaining two sites on gp120 and one on gp41 were distinguished by competition binding assays but could not be defined by overlapping peptides, suggesting that they were discontinuous or conformational epitopes. Five of the 28 MAbs consistently and reliably neutralized the infectivity of SIVmac251. Two of these bound to a peptide (aa171-190) in the V2 region. The remaining three MAbs bound to a conformational epitope on gp120. These two neutralizing epitopes on SIV are analogous to similar epitopes recently described in HIV-1. In contrast, three MAbs binding to the V3 region of SIV failed to neutralize infectivity, suggesting that this region in SIV may by functionally different from the V3 loop in HIV-1.