The UL69 open reading frame of human cytomegalovirus (HCMV) is homologous to the immediate-early protein ICP27 of herpes simplex virus, an essential viral regulatory protein involved in the transition from early to late gene expression. Genes with homology to ICP27 have been detected in all subclasses of herpesviruses so far. While the respective proteins in alpha- and gammaherpesviruses have been defined as trans-regulatory molecules, nothing is known about these genes in betaherpesviruses. This study was therefore undertaken in order to investigate expression from the UL69 gene locus of HCMV. Northern (RNA) blot experiments revealed a complex pattern of transcripts that changed during the time course of the HCMV replicative cycle: two transcripts of 2.7 and 3.5 kb that were regulated differentially could be detected as early as 7 h after infection. However, these transcripts could not be detected in the presence of cycloheximide. Additional, larger transcripts were present exclusively at late times after infection. To analyze protein expression from the UL69 gene region, the UL69 open reading frame was expressed as a histidine-tagged protein in Escherichia coli. A specific antiserum was generated and used to detect the UL69 protein in HCMV-infected cells which revealed its localization within the intranuclear inclusions that are characteristic for HCMV infection. In cotransfection experiments, an HCMV true late promoter could not be activated by UL69, whereas an early promoter and several heterologous promoters were stimulated about 10-fold. Complementation studies showed that the UL69 protein cannot substitute for ICP27 in the context of the HSV infection, suggesting functional differences between these two proteins. In summary, these experiments define a novel regulatory protein encoded by HCMV that is expressed as an early-late gene and appears to exert a broad stimulatory effect on gene expression.