Previous studies have shown that infection of G0-synchronized human fibroblasts by human cytomegalovirus (HCMV) results in a block to cellular DNA synthesis. In this study, we have examined the effect of viral infection on the formation of the host cell DNA prereplication complex (pre-RC). We found that the Cdc6 protein level was significantly upregulated in the virus-infected cells and that there was a delay in the expression of the Mcm family of proteins. The loading of the Mcm proteins onto the DNA pre-RC complex also appeared to be defective in the virus-infected cells. This inhibition of DNA replication licensing was associated with the accumulation of geminin, a replication inhibitor. Cdt1, which participates in the loading of the Mcm proteins, was also downregulated and modified differentially in the infected cells. Early viral gene expression was sufficient for the virus-induced alteration of the pre-RC, and the immediate-early protein IE1 was not required. These studies show that the inhibition of replication licensing in HCMV-infected cells is one of the multiple pathways by which the virus dysregulates the host cell cycle.