To evaluate the usefulness of a human T-cell lymphotropic virus type I (HTLV-I) recombinant p21E immunoassay as a supplementary test in HTLV-I/II serologic testing algorithms, we used this assay to test 378 serum samples previously categorized as positive, indeterminant, or negative for HTLV-I/II antibody, as defined by U.S. Public Health Service guidelines. We found this test to be highly sensitive for detecting antibody to HTLV-I/II env (99.4%) but slightly less specific (96.0%), particularly among samples from intravenous drug users. Our data suggest that this test is most appropriately used to confirm the absence of env antibody in samples which are repeatably reactive in an HTLV-I/II screening assay and gag reactive only by immunoblotting. Because of the high sensitivity of this recombinant p21E test, a negative result in this context could preclude radioimmunoprecipitation testing. However, pending further evaluation of the specificity of this assay, samples testing positive for p21 env antibody may require confirmation by radioimmunoprecipitation, particularly in situations in which the results will be used for diagnostic purposes or blood donor counseling.