Two different approaches were used to map the type-specific regions on human T cell leukemia virus (HTLV) envelope glycoproteins. 1) Antibody reactivities of polymerase chain reaction-confirmed HTLV-I or HTLV-II carriers' sera were analyzed by Western blot assay with seven recombinant proteins containing different regions of HTLV-I or HTLV-II envelope proteins. 2) Rabbit antibodies elicited by nine HTLV-I Env synthetic peptides were used to react with the native HTLV envelope glycoproteins in an antibody-dependent cellular cytotoxicity (ADCC) assay. The results of the Western blot analysis showed that RP-B2, which contains amino acid residues 166 to 213 from HTLV-II exterior glycoprotein, was specifically reactive with 90.6% (48 of 53) of the HTLV-II carriers' sera but not with any of the HTLV-I carriers' serum (0 of 71). In contrast, RP-B, which contains amino acid residues 166 to 229 from HTLV-I exterior glycoprotein, was reactive with 85.1% (114 of 134) of the HTLV-I carriers' sera but not with any HTLV-II carrier serum (0 of 62). Furthermore, anti-HTLV-I Env synthetic peptide antibody-mediated ADCC identified several distinguishing HTLV-I ADCC epitopes in the middle region (amino acid residues 177 to 257) of the HTLV-I exterior glycoprotein. Therefore, HTLV type-specific epitopes reside mainly in a 69-amino acid sequence bounded by two cysteine residues (amino acids 157 and 225 for HTLV-I and 153 and 221 for HTLV-II), in the middle region of the exterior envelope glycoproteins.