Early diagnosis of acute human immunodeficiency virus (HIV) infection is difficult because patients may be seronegative for HIV at the time of presentation. We have used a serum HIV antigen (HIV-Ag) enzyme immunoassay (EIA) to diagnose acute HIV infection in four high-risk patients. The clinical syndrome in these four patients was characterized by fever (four), rash (three), myalgias-arthralgias (three), and pharyngitis (two). All patients had spontaneous resolution of their symptoms within eight to 12 days. Serum HIV antibody, as measured by a commercially available screening EIA and by Western blot analysis, was negative in all patients at time of presentation and all seroconverted on subsequent testing. Human immunodeficiency virus was isolated from two of two patients during the acute illness. Initial serum samples from all four patients were positive for HIV-Ag. Serum samples of three of four patients became negative for HIV-Ag and positive for HIV antibody. These data suggest that serum HIV-Ag detection by EIA may be useful in the diagnosis of the acute syndrome caused by HIV infection.