Repression of human cytomegalovirus alpha (immediate-early) gene expression is under the control of the viral ie2 gene. Here we show that ie2 negatively regulates gene expression directed by the strong cytomegalovirus enhancer via a specific 15-bp target sequence (which we term cis repression signal [crs]). This crs is located between -14 and +1 relative to the transcription start site and will function in an orientation-independent fashion, consistent with repression occurring at the transcriptional level. Repression is dominant over transactivation by ie1 gene products. The crs (5'-CGTTTAGTGAACCGT-3') does not contain previously recognized binding sites for cellular transcription factors, and a precise copy is not found elsewhere in the human cytomegalovirus genome. The position of the signal near the transcription start site appears to be important in function; addition of the crs near the transcription start site of a heterologous promoter, from the thymidine kinase gene of herpes simplex virus type 1, conferred cytomegalovirus ie2-dependent repression upon this promoter. Thus, we propose that an ie2 gene product or an induced cellular protein mediates repression by binding to crs. Negative regulation of alpha gene expression may be important during viral replication or latency.