Latent cytomegalovirus (CMV) infection is often reactivated in the lung. We postulated that this reactivation could occur by stimulation of the CMV major immediate early (IE) promoter by other viruses that infect the lung. The specific aim of this study was to investigate whether adenovirus early proteins could stimulate the CMV IE promoter in inflammatory cells. We transfected the monocyte/macrophage THP-1 cell line and the T-lymphocyte Jurkat cell line with plasmids coding for adenovirus E1A 12S or 13S proteins, along with a plasmid containing the CMV IE promoter region linked to the chloramphenicol acetyltransferase (CAT) reporter gene. In unstimulated THP-1 cells, the E1A 13S gene product increased CMV IE CAT activity by 18-fold compared with cells containing the control E1A plasmid. This effect was not seen in cells transfected with the E1A 12S plasmid. There was a similar effect of the E1A 13S gene product in LPS-stimulated THP-1 cells. In unstimulated Jurkat cells, the E1A 13S gene product stimulated CMV IE CAT activity by 19-fold compared with cells containing the E1A control plasmid; the E1A 12S gene product had no effect. There was a similar effect of the 13S E1A gene product in phorbol myristate acetate-stimulated Jurkat cells. These findings demonstrate that the CMV IE promoter can be stimulated by early viral proteins of adenovirus in inflammatory cells. These observations could be important for understanding the reactivation of latent CMV infection.