The activation of the major immediate-early promoter (MIEP) is a key event in the cytomegalovirus replication cycle and is dependent on cellular transcription factors which are partially activated by viral proteins. Expression of the viral chemokine receptor homolog US28 results in constitutive activation of pro-inflammatory transcription factors that may be involved in the activation of the major immediate-early promoter/enhancer. Using reporter gene assays in human embryonic kidney cells, we found that US28 signaling was responsible for increased major immediate-early promoter/enhancer activity which was independent of beta-chemokine binding. Inhibition of nuclear factor-kappaB (NF-kappaB) only partially blocked the effect of US28, whereas treatment with a specific p38 mitogen activated kinase (MAPK) inhibitor fully abrogated the US28-induced enhancement of promoter activity. Our results suggest that during human cytomegalovirus (HCMV) infection, US28 in epithelial cells transactivates the major immediate-early promoter/enhancer via the activation of p38 MAPK and downstream signaling that partially involves NF-kappaB.