Reactivation of Epstein-Barr virus (EBV) in early human immunodeficiency virus (HIV) infection was investigated in 49 homosexual men who seroconverted to HIV (cases) as compared with 49 matched controls who remained seronegative to HIV during a longitudinal study. EBV infection was reactivated in cases 6 months, but not 12 months, prior to HIV seroconversion as compared with controls and remained reactivated during 18 months of follow-up after HIV seroconversion, as shown by increases in immunoglobulin (Ig) G antibody titers to EBV early antigen. Antibody titers to EBV viral capsid antigen did not differ between cases and controls prior to the time of seroconversion to HIV but were significantly increased among cases by the first seropositive study visit and remained elevated during the 18 months after HIV seroconversion. Total serum IgG levels were increased in cases at the visit of seroconversion, and during 18 months of follow-up, but did not correlate with enhanced IgG production specific for EBV antigens. Significant decreases in numbers of CD4+ cells and increases in numbers of CD8+ cells during this early phase of HIV infection were not associated with changes in patterns of EBV antibody responses. Reactivation of EBV beginning 6 months before HIV seroconversion may have implications regarding the role of this herpesvirus in the pathogenesis of HIV.