The human T lymphotropic viruses (HTLV-I and -II) are relatively common in subpopulations of certain countries, notably intravenous drug abusers in North America. Infections with these malignancy-associated human retroviruses are hard to discriminate with currently available commercial serological tests. We studied the distribution of antigenicity and the degree of cross-reactivity of epitopes in gag and env of the two viruses. Sequences in the carboxyl terminus of the matrix protein (MA) and the middle of the outer glycoprotein (SU) reacted in a type-specific fashion, while sequences from the capsid protein (CA), the carboxyl terminus of SU, and conserved portions of the transmembrane protein (TM) mainly reacted in a group-specific fashion, correlating with the degree of sequence dissimilarity between the two viruses. The serological discrimination obtained with the peptides was evaluated in a panel of 25 sera where infection with HTLV-I or -II had been typed by competition in a p24 enzyme-linked immunosorbent assay (ELISA) or by the polymerase chain reaction (PCR). After processing peptide results in a computer program, a typing result concordant with earlier results was obtained in 21 of 25 sera. Of the remaining five sera, four were labeled "too weak for typing" and one "HTLV of uncertain type" by the program. They did not react sufficiently strongly or clearly with the peptides to allow classification. A combination of synthetic peptides may become useful for serotyping HTLV infection and become an alternative to Western blots for confirmation of HTLV positivity.