The human cytomegalovirus (HCMV) major immediate-early (MIE) promoter has strong transcriptional promoting capability. Its cis-acting regulatory elements form a special structure in this region that is repeated multiple times; the biological significance of these elements and their different compositions in the transcriptional promoting process remain unclear. Our results demonstrate that the HSV-I MIE protein ICP22 can generate strong repression of many viral and cellular promoters and enhancers. We further studied the transcriptional effects of ICP22 on structural elements and mutations in various HCMV MIE promoters by using a CAT assay. In spite of different transcriptional effects of all the elements in the presence of ICP22, the transcriptional efficiencies exhibited by mutations generated by different compositions and an entire HCMV promoter, are not the simple sum of the functions of these elements. Furthermore, the transcriptional activities of specific sequences were not affected by the presence of ICP22. Therefore, it is assumed that the HCMV MIE promoter co-regulates expression of downstream genes by using viral and cellular specific factors via a specific pathway.