The third member of the human T-cell leukemia (lymphotrophic) retrovirus family (HTLV-III) is a newly discovered retrovirus that has been closely associated with the acquired immunodeficiency syndrome (AIDS). In our application of an enzyme-linked immunosorbent assay (ELISA) for HTLV-III antibodies, 72 (82%) of 88 patients with AIDS were positive, 14 (16%) were borderline, and two (2%) were negative. In contrast, only 1% of 297 volunteer blood donors were positive, 6% were borderline, and 93% were negative, demonstrating that this ELISA for HTLV-III antibodies is highly specific and sensitive for AIDS (excluding borderline results, 98.6% and 97.3%, respectively). Among persons at high risk for AIDS, 8% had borderline results, with positive and negative results readily distinguished as bimodal distributions that paralleled the temporal and geographic trends in AIDS. None of the 188 laboratory and health care employees working with patients with AIDS or their specimens were positive for HTLV-III antibodies, indicating that current precautions for health care workers appear adequate. This ELISA for HTLV-III antibodies will be a useful screening test among blood donors and populations at risk for AIDS, will aid in the diagnosis of suspected AIDS, and will help in defining the spectrum of diseases that are etiologically related to HTLV-III.