A synthetic-peptide-based enzyme-linked immunosorbent assay (EIA) capable of screening for antibodies to both human immunodeficiency virus type 1 (HIV-1) and HIV-2 has been developed for use in blood banks and diagnostic laboratories. Microtiter wells are coated with two synthetic peptides, one corresponding to the highly conserved envelope region of HIV-1 and another corresponding to the conserved envelope region of HIV-2. Overall, sensitivity was 100% in 303 individuals diagnosed with AIDS and 96 individuals diagnosed with AIDSrelated complex, 14.8% in a study of 500 high-risk group members, 99.9% in 600 EIA repeatedly reactive (RR)-HIV-1 Western blot (WB)-positive repository specimens, and 100% for 222 geographically diverse HIV-1 specimens and 216 confirmed HIV-2-positive specimens evaluated. The specificity was determined to be 99.72% for a total of 13,004 serum and plasma samples from random volunteer donors evaluated across five blood banks. Forty donors who were found to be EIA RR-WB indeterminate but nonreactive on the United Biomedical, Inc., test (UBI HIV 1/2 EIA) were prospectively followed as an additional measure of specificity. None of the 40 low-risk cases evolved into a positive WB pattern at follow-up. The sensitivity and specificity of this new assay are comparable to those of other Food and Drug Administration-licensed HIV-1 and HIV-1-HIV-2 assays that are currently available in the United States. The UBI HIV 1/2 EIA affords laboratories another choice in the detection of antibodies for HIV-1 and HIV-2 with a test based on an alternative antigen format.