Several cis-acting transcriptional silencer elements that are dispersed over a long (more than 1 kb) region upstream of the mouse DNA polymerase-beta gene repress transcription not only of this gene but also of heterologous promoter-enhancers of other genes. The effect of DNA replication on the functions of these silencer elements was examined by combining them with transiently replicating plasmids carrying the chloramphenicol acetyltransferase (CAT) gene and the replication origins (ori) of polyoma virus and SV40 DNA. The silencers functioned efficiently when these plasmids were transfected into mouse NIH 3T3 and monkey CV1 cells. These cells lack T antigen and therefore do not allow replication of the plasmids. The silencers also functioned when the plasmids contained nonfunctional SV40 ori. In contrast, the negative effect of the silencer was not observed when the plasmids were transfected into cells producing large T antigens, such as MOP8 cells and COS1 cells. The observed differences in the silencer function between the permissive and nonpermissive cells was not due to differences in plasmid copy numbers in the cells. The results indicate that ongoing DNA replication can completely overcome the negative effects of the silencer elements on transcription.