Individuals infected with the human immunodeficiency virus (HIV), the etiologic agent of acquired immunodeficiency syndrome (AIDS), often show symptoms associated with reactivation of Epstein-Barr virus (EBV). In this study, we show that exposure of EBV-positive B lymphocytes to HIV-1 in vitro induced the EBV replicative cycle in these cells, as evidenced by an increased proportion of cells expressing EBV early antigens (EA) and capsid antigens (VCA). Reactivation of EBV by HIV-1 appeared to be virus-dose-dependent and required virus penetration and expression in B cells. Although HIV-1 RNA was detected by in situ hybridization in the majority of HIV-1-infected B lymphocytes, induction of EA and VCA was transient and limited to less than 20% of the cell population. The tumor-promoting phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and HIV-1 acted synergistically and had similar kinetics in inducing the expression of EBV. Direct reactivation of EBV by HIV-1 may contribute to the role of EBV as a factor in the genesis of AIDS-related conditions.