Human herpesvirus 6 (HHV‐6) and human immunodeficiency virus type 1 (HIV‐1) may interact during transplacental transmission of HIV‐1. The placental syncytiotrophoblast layer serves as the first line of defense of the fetus against viruses. Patterns of replication of HHV‐6 variant A (HHV‐6A) and HIV‐1 were analyzed in singly and dually infected human term syncytiotrophoblast cells cultured in vitro. For this purpose, the GS strain of HHV‐6A and the Ba‐L and IIIB strains of HIV‐1 were used. HHV‐6A replication was restricted at the level of early gene products in singly infected syncytiotrophoblasts, whereas no viral protein expression was found in cells infected with HIV‐1 alone. Coinfection of syncytiotrophoblast cells with HHV‐6A and HIV‐1 resulted in production of infectious HIV‐1. In contrast, no enhancement of HHV‐6A expression was observed in cell cultures infected with both viruses. Uninfected syncytiotrophoblast cells were found to express CXCR4 and CCR3 but not CD4 or CCR5 receptors. Infection of syncytiotrophoblasts with HHV‐6A did not induce CD4 expression and had no influence on chemokine receptor expression. Activation of HIV‐1 from latency in coinfected cells was mediated by the immediate‐early (IE)‐A and IE‐B gene products of HHV‐6A. Open reading frames U86 and U89 of the IE‐A region were able to activate HIV‐1 replication in a synergistic manner. The data suggest that in vivo double infection of syncytiotrophoblast cells with HHV‐6A and HIV‐1 could contribute to the transplacental transmission of HIV‐1 but not HHV‐6A. J. Med. Virol. 67:67–87, 2002. © 2002 Wiley‐Liss, Inc.