A sensitive and specific avidin-biotin enzyme immunoassay that uses immunoreagents from naturally infected individuals was formulated for the detection of human immunodeficiency virus (HIV) antigens. A total of 500 cell culture samples from 111 cultures were tested in this assay and in a reverse transcriptase (RT) assay. Of 353 samples that were positive in the immunoassay, 174 were positive and 179 were negative in the RT assay. The specificity of the immunoassay results was supported by the failure of samples to react with nonimmune serum, by the ability of an anti-HIV type 1 monoclonal antibody to block the reactivity of selected samples, and by the appearance of RT activity in samples drawn from some cultures after a longer period of cultivation. HIV antigens were detected in 174 of 176 RT-positive samples (sensitivity, 98.9%). A comparison of the kinetics of antigen production and RT activity revealed that detectable antigen levels frequently preceded the appearance of RT activity. Thus, 50% of virus-containing cultures were identified within 9 days by immunoassay compared with 14 days by RT assay. In addition, RT activity was often detected intermittently in cultures sampled on several days, whereas antigen levels did not decline after initial appearance. Enzyme immunoassays for HIV antigen detection that use easily obtained reagents and simple technologies could facilitate laboratory and clinical research which requires cultivation of viruses.