Synthetic peptides corresponding with unique regions of the envelope glycoproteins (gp46) of human T cell lymphotropic viruses (HTLVs) were used in an enzyme immunoassay to determine if HTLV-I and -II infections could be discriminated. Two synthetic HTLV-I sequence-derived peptides, Env-1 (amino acids 191-215) and Env-5 (amino acids 242-257), reacted with 92% and 100% of the serum specimens (n = 52) from HTLV-I-infected persons, respectively. Although a small percentage (8.6%) of serum specimens from persons infected with HTLV-II cross-reacted with Env-1, none of these specimens reacted with Env-5. Peptide Env-2 encoded by the envelope region of HTLV-II (amino acids 187-210) reacted with serum specimens from both HTLV-I (94%)- and HTLV-II (74%)-infected patients, whereas Env-6, another HTLV-II peptide (amino acids 238-254), reacted with less than 6% of the specimens. Therefore, the Env-5 peptide with amino acid sequence SerProAsnValSerValProSerSerSerSerThrProLeuLeuTyr represents an immunodominant domain of HTLV-I that is recognized by serum antibodies from all HTLV-I-infected persons. Moreover, the Env-5-based ELISA allows a categorical distinction between the closely related HTLV-I and -II infections.