Dual cis-acting silencer elements are located upstream of the mouse DNA polymerase beta gene (Yamaguchi, M., Hayashi, Y., and Matsukage, A. (1989) J. Biochem. (Tokyo) 105, 79-83). In order to examine possible involvement of transcriptional silencer elements in the regulation of DNA replication, we have utilized a transient replication system of the plasmid DNA carrying replication origin of polyoma virus DNA in mouse MOP8 cells, which is constitutively producing polyoma virus large T-antigen. The polyoma virus origin of DNA replication is composed of three cis-acting genetic elements called alpha, beta, and core, in which alpha and beta elements correspond to enhancer domains. When the 5'-flanking regions of the DNA polymerase beta gene containing silencer elements were placed at the late gene border of alpha element, they effectively repressed the DNA replication. However, when placed at the early gene border of core element, it only marginally repressed the DNA replication. These results suggest that the silencer elements at cis position repress polyoma virus DNA replication by impeding the enhancer function that activates the DNA replication.