The human malaria parasite, Plasmodium falciparum, maintains a persistent infection altering the proteins expressed on the surface of the infected red blood cells, thus avoiding the host immune response. The primary surface antigen, a protein called PfEMP1, is encoded by a multicopy gene family called var. Each individual parasite only expresses a single var gene at a time, maintaining all other members of the family in a transcriptionally silent state. Previous work using reporter genes in transiently transfected plasmid constructs implicated a conserved intron found in all var genes in the silencing process. Here we have utilized episomal recombination within stably transformed parasites to generate different var promoter and intron arrangements and show that loss of the intron results in var promoter activation. Further, in multicopy plasmid concatamers, each intron could only silence a single promoter, suggesting a one-to-one pairing requirement for silencing. Transcriptionally active, "unpaired" promoters remained active after integration into a chromosome; however, they were not recognized by the pathway that maintains mutually exclusive var gene expression. The data indicate that intron/promoter pairing is responsible for silencing each individual var gene and that disruption of silencing of one gene does not affect the transcriptional activity of neighboring var promoters. This suggests that silencing is regulated at the level of individual genes rather than by assembly of silent chromatin throughout a chromosomal region, thus providing a possible explanation of how a var gene can be maintained in a silent state while the immediately adjacent var gene is transcriptionally active.