Valproic acid (VPA), a simple branched-chain fatty acid having anticonvulsant activity and used in the treatment of many forms of epilepsy, markedly stimulated human cytomegalovirus (HCMV) replication in human fibroblasts (MRC-5 cells). The maximum level of stimulation was reached when cells were treated for 24 h before infection. The enhancement of virus replication correlated with an increase in the number of immediate early (IE) and early (E) antigen-positive cells. VPA also induced expression of IE antigens after transfection of fibroblasts with a plasmid containing the entire IE1-2 region. Moreover, VPA stimulated the HCMV IE1-2 promoter/enhancer-mediated expression of beta-galactosidase in a stably transfected Jurkat T cell line. Recently, VPA was shown to inhibit glutathione reductase in human red blood cells, but an action through the glutathione metabolic pathway can be eliminated in this case, since VPA decreased the intracellular level of glutathione in Jurkat T cells but not in MRC-5 cells. The ability of VPA to stimulate HCMV replication provides an attractive model for studying the molecular mechanism of the regulation of HCMV IE1-2 gene expression.