We have evaluated a new serological confirmatory test (INNO-LIA HTLV I/II Ab [INNO-LIA]) for human T-cell leukemia virus (HTLV) using a large collection of samples from Brazilian blood donors (São Paulo region) and compared the results with those obtained by Western blotting (WB) tests (WB2.3 and WB2.4). Blood donations were initially screened by enzyme-linked immunosorbent assays (ELISAs) based on viral lysates, and repeatedly reactive samples were further tested by WB2.3. When available, samples were also tested by PCR, two additional ELISAs based on recombinant antigens (recombinant ELISAs), a new-generation WB assay (WB2.4), and the INNO-LIA. Of the 18,169 samples tested, 292 (1.61%) were repeatedly reactive in the ELISAs (viral lysate based) and were further tested by WB2.3; 97 were positive (19 that were typed as HTLV type I [HTLV-I], 12 that were typed as HTLV type II [HTLV-II], and 66 that were nontypeable), 17 were negative, and 178 had indeterminate results. Of the samples with indeterminate results, 172 were tested by INNO-LIA, which could resolve 153 samples as negative. Regarding the positive samples, WB2.3 and INNO-LIA produced concordant results for all HTLV-I-positive samples, whereas for HTLV-II they agreed for 10 of 12 samples; the 2 samples with discordant results were considered to be positive for HTLV-II by WB with WB2.3 but negative for HTLV-II by INNO-LIA and the two recombinant ELISAs. Furthermore, of the 66 nontypeable samples, 60 underwent testing by INNO-LIA; 54 turned out to be negative by the latter test as well as by recombinant ELISAs. In conclusion, the new serological confirmatory assay for HTLV (INNO-LIA HTLV I/II Ab) resolved the results for the majority of the indeterminate and positive-untypeable samples frequently observed by WB assays.