An assay system was developed for the analysis of antibodies secreted in vitro against human immunodeficiency virus (HIV) by cultured peripheral blood lymphocytes of HIV-infected individuals. Cultures of peripheral blood lymphocytes were established with medium alone or with medium containing Epstein-Barr virus (EBV) or pokeweed mitogen. HIV antibodies were determined by an ELISA performed with commercial kits in which a whole virus extract served as antigen. Optimal antibody secretion was detected in 7-day peripheral blood lymphocyte cultures to which EBV had been added to provide polyclonal B-cell activation. Pokeweed mitogen-induced antibody secretion and spontaneous antibody secretion were less consistent. With EBV as a stimulus, the sensitivity and specificity of this assay for determining HIV infection status were each 100% in adults. When the assay was applied to infants and children, 23 of 24 symptomatic HIV-seropositive children (class P-2) and 11 of 33 asymptomatic seropositive infants aged less than or equal to 15 months (class P-0) tested positive for EBV-induced antibody secretion. Six of the 11 P-0 patients who tested positive have progressed to develop symptomatic disease, while the remainder are still seropositive at ages 2-15 months. Of the infants who were negative in this assay, all have remained asymptomatic. Treatment with 3'-azido-3'-deoxythymidine in infected adults and children has resulted in transient suppression of the in vitro antibody response in some instances. Thus EBV-induced synthesis of HIV-specific antibodies in vitro is a sensitive and specific indicator of HIV infection and is of help in determining infection status of asymptomatic seropositive infants who are classified as having "indeterminate" infection.