Current algorithms for the serologic confirmation of human T-cell lymphotropic virus type I and II (HTLV-I/II) antibody reactivity are complicated. We evaluated the performance of an HTLV-I Western blot (immunoblot) spiked with recombinant p21e protein (p21e WB) as an alternative to current confirmatory methods. These methods include the HTLV-I viral lysate Western blot and either a radioimmunoprecipitation assay or a p21e enzyme-linked immunosorbent assay. Five hundred fifty nine blood donations obtained from five U.S. blood centers and classified as HTLV-I/II seropositive (n = 149) or seroindeterminate (n = 410) by routine testing methods were further evaluated by PCR for proviral DNA and by the p21e WB. On the basis of serologic and PCR testing, 155 donations were classified as HTLV-I/II infected. The sensitivity of the p21e WB was 97.4%, slightly exceeding that of routine confirmatory testing. The specificity of the p21e WB was 97.5%, as determined by testing of 404 seroindeterminate samples that were negative in the PCR. The positive predictive value of the p21e WB was 94%. In contrast, the specificity and positive predictive value of routine confirmatory testing were both 100%. Follow-up sampling of presumptive p21e WB false-positive donors substantiated the absence of HTLV-I/II infection. Although the p21e WB used in this study has high sensitivity and may be useful as a confirmatory assay in epidemiologic research studies, it may not be ideal as a confirmatory test for the notification of blood donors.