Having observed that multiple neutralization epitopes of human T-cell-leukemia-virus-type-l (HTLV-I) envelope gp46 clustered in a region between amino acids 187 and 199, we speculated that a region of HTLV-II gp46 corresponding to the HTLV-I-neutralization region may contain HTLV-ll-specific neutralization epitopes. To test this, we immunized a NZW rabbit and BALB/c mice with a synthetic peptide containing the HTLV-II gp46 amino acids I82 to I99 (pep 182-199) conjugated to OVA. The serum from the rabbit reacted to the HTLV-II gp46 and neutralized HTLV-ll-mediated syncytium formation. One monoclonal antibody (MAb), M2E I86N I, generated from the BALB/c mice, stained specifically the surface of HTLV-IIpositive cells and reacted with HTLV-II gp46 by Western blot. This MAb was of the IgA isotype and inhibited HTLV-II-but not HTLV-I-mediated syncytium formation at a final antibody concentration of 200 pg/ml; it also neutralized an HTLV-II-VSV pseudotype virus specifically. Epitope mapping by ELISA using overlapping synthetic peptides indicated that the neutralization epitope recognized by the M2E186NI MAb was located between HTLV-ll gp46 amino acids 186 and 192, corresponding to the sequence Leu-Gln-His-Val-lle-Leu-Gln-Pro. These new observations will be helpful in developing vaccines against HTLV-II infection.