This investigation compares the results of a new method of diagnosing HIV-1 infection in infants < 6 months of age with currently employed techniques including cocultivation, the polymerase chain reaction (PCR), serum p24 antigen, and in vitro antibody production (IVAP) measurements. The new method, called in vitro antigen (IVAG), measures p24 antigen released into culture supernatants of peripheral blood mononuclear cells that are incubated with Epstein-Barr virus (EBV). No activated donor lymphocytes or interleukin 2 (IL-2) are added to the culture. Using this technique, HIV-1 infection was detected in 15 of 17 HIV-1-infected infants < 2 months of age, including 3 of 7 infants tested at birth, and 15 of 15 HIV-1-infected infants between 2 and 6 months of age. None of 83 determinations of 15 uninfected infants were positive. These results were found to be comparable to results obtained by the traditional cocultivation technique and the polymerase chain reaction. Because of its simplicity and reduced cost, this sensitive and specific assay could be a valuable addition to the current methods of diagnosis of HIV-1 infection in young infants.