To detect human immunodeficiency virus (HIV) antibodies in a simple enzyme-linked immunoassay (CBre3-EIA), we used an Escherichia coli-expressed polypeptide antigen, representing the carboxy-terminal third of the external membrane glycoprotein gene fused with the amino-terminal half of the transmembrane glycoprotein gene. Over a 3-month period, 2707 consecutive serum samples referred for confirmatory testing for human T-lymphotrophic virus type III (HTLV-III) antibodies were evaluated by both Western blot and CBre3-EIA. On a single determination for each sample, the CBre3-EIA was found to have an estimated sensitivity (99.9%) and specificity (99.1%) similar or superior to the more cumbersome Western blot method. This study shows that all HIV-seropositive subjects have antibodies to the virus envelope protein; no other virus antigens are required for construction of highly sensitive immunoassays.