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Objective To evaluate the effect of dimethyl sulfoxide (DMSO) on the major immediate early promoter of human cytomegalovirus. Methods THP-1 cells were transfected with plasmids pCAT760, pIE1-163, pIE1-183, pIE1-193 and pIE1-213, respectively, and exposed to dimethyl sulfoxide in the presence or absence of lipopolysaccharide. The extracts of these cells were performed by chloramphenicol acetyl transferase assay. The nuclear extracts of THP-1 cells treated with dimethyl sulfoxide in the presence or absence of lipopolysaccharide were prepared for the DNA binding reaction by electrophoretic mobility shift assay.Results Dimethyl sulfoxide increases the expression of the cytomegalovirus major immediate early promoter, and also upregulated expression of the minimal major immediate early promoter containing triplicates of either the 18 base pair or 19 bsae pair sequences. Furthermore, gel mobility shift assays showed that dimethyl sulfoxide enhances Nuclear Factor κB and CyclicAMP response element binding protein to bind to the 18 base pair and 19 base pair repeats.Conclusions One mechanism whereby DMSO enhances human cytomegalovirus replication is through upregulating the major immediate early promoter. This effect on human cytomegalovirus gene expression is, in part, related to enhanced Nuclear Factor κB and CyclicAMP response element binding protein activity.