Human cytomegalovirus (HCMV) infection of fibroblasts induces the proinflammatory mediator macrophage migration inhibitory factor (MIF). Our in vitro experiments show that active HCMV infection alone is required to induce an early and sustained induction of MIF mRNA and protein production. Unlike in other infection models, in which MIF has been described to be released from preformed stores, our data conclusively show that HCMV infection triggers de novo synthesis and subsequent secretion of MIF. The kinetics of MIF protein production during HCMV infection points to an efficacious immune modulation, in which lymphocytes and monocytes are initially recruited by the early release of chemokines to sites of infection and locally activated by increasing concentrations of MIF.