The influence of DMA methylation in vitro on the activity of the very strong human cytomegalovirus (HCMV) major immediate early (IE) modulator/enhancer/promoter region was investigated by transient transfection experiments of premonocytic HL-60 cells. While sequence-specific methylation of the major IE enhancer and/or modulator with the cytosine methyltransferases FnuDII, Hhal and Haelll had no significant effect, the promoter activity was completely repressed by methylation of the cytosine in 5'-CpG sites with the Spiroplasma methyltransferase Sssl. Addition of TNF-α or PMA which are strong stimulators of HCMV major IE enhancer/promoter activity in premonocytic HL-60 cells had no effect on repression. Inactivation of the IE enhancer/promoter via methylation by M. SssI could be partially alleviated by co-transfection with an excess of untranscribable highly methylated DNA. These results indicate that a methyl-CpG binding factor is involved as mediator in the inhibitory effect of HCMV enhancer/promoter methylation. Taken together, the HCMV major IE enhancer/promoter has been shown to be susceptible to transcriptional inactivation by methylation of the cytosines in CpG dinucleotides, a process that is proposed to play a modulatory role in viral latency.