The principle route of acquisition of cytomegalovirus (CMV) for the fetus is believed to be via the placenta. We subjected purified cytotrophoblast cells obtained from full-term placentas to CMV infection and examined placental gene expression using microarray analyses. Cytotrophoblast cells purified from term placentas differentiated in vitro into a multinucleated syncytium that could be productively infected with CMV, with peak virus titers of approximately 10 plaque-forming units (PFU)/mL identified in supernatants at late time points postinoculation. Infected syncytiotrophoblast cells expressed CMV-specific transcripts and proteins, as demonstrated by Northern blot and immunofluorescence assays. Microarray analyses revealed that CMV infection strongly and reproducibly altered trophoblast gene expression, elevating expression of mitotic cell cycle genes, and repressing expression of genes associated with trophoblast differentiation, particularly those associated with formation and stabilization of the extracellular matrix. We conclude that purified, differentiated syncytiotrophoblasts are permissive for CMV replication. Infection of these cells induces significant perturbations in trophoblast transcription. An improved understanding of the molecular events that occur during CMV infection of trophoblasts could provide insights into interventions that might prevent or minimize congenital transmission.