The present study evaluated a new confirmatory assay for antibodies to human T-cell lymphotropic virus type 1 and 2 (HTLV-1 and HTLV-2) proteins performed with serum samples from various commercial sources. The new test is a line immunoassay (LIA) with a nylon membrane sensitized with the most relevant antigens of HTLVs: the envelope gp46 and gp21 as well as the gag p24 and p19 antigens, represented by either recombinant proteins or synthetic peptides. A total of 176 serum or plasma samples were tested, of which 66 were HTLV-1 positive, 72 were HTLV-2 positive, and 38 were HTLV negative; of the 38 HTLV-negative samples 23 were indeterminate by Western blotting (WB). Serially diluted samples (n = 33) from HTLV-1- and HTLV-2-infected patients were also analyzed to determine the sensitivity of the new assay. The new confirmatory assay (INNO-LIA HTLV) performed markedly better than WB assays for those samples reactive by screening. Accurate confirmation of the presence of HTLV-1 and HTLV-2 antibodies and accurate discrimination of HTLV-1 and HTLV-2 antibodies were obtained for all the HTLV-seropositive samples. Due to its enhanced specificity and sensitivity, the new assay not only improves the ability to confirm and discriminate HTLV infections but also eliminates the vast majority of WB-indeterminate and false-positive specimens.