Abstract: Method and assay kit for positively identifying HTLV-I and HTLV-II infection from human serum samples. The kit includes peptide antigens from the C-terminal regions of HLTV-I p19 and HTLV-II p21 gag proteins, and peptide antigens from the HLTV-I and HTLV-II env proteins immobilized on a solid support. After reaction of the serum sample with the solid support, an antibody-detection reagent in the kit is added to the support, to detect binding of human serum antibodies to each of the peptide antigens separately. The test allows positive identification of HTLV-I or HTLV-II when antibody binding to each HTLV-I or HTLV-II gag and env peptide antigen, respectively, is observed. Also disclosed is a kit for screening human sera for evidence of HTLV-I or HTLV-II infection.