With the cloning of DNA encoding the trans -dominant negative mutant form of the HSV-1 origin-binding protein UL9, UL9-C535C, under the control of the tet operator-bearing hCMV major immediate-early promoter (pcmvtetO), this article demonstrates that the tetR-mediated mammalian transcription repression switch (Yao et al., Hum. Gene Ther. 9:1939-1950, 1998) can be converted to a novel HSV-1-specific viral replication switch. Using this viral replication switch, the plaque-forming efficiency of infectious HSV-1 DNA can be reversibly regulated by tetR over 100-fold in transient viral infection assays. Moreover, while less than 0 PFU/ml of HSV1 was detected from tetR-expressing cells transfected with infectious HSV-1 DNA and plasmid pcmvtetOUL9-C535C in the presence of tetracycline, close to 1000 PFU/ml of HSV-1 was produced when similar experiments were carried out in the absence of tetracycline. The tetracycline treatment led no reduction in HSV-1 synthesis in cells transfected with infectious HSV-1 DNA alone. Taken together, given that the UL9-C535Cassociated antiviral activity can be silenced in the context of this HSV-1 replication switch, the establishment of this reversible switch would allow construction of a new generation of HSV-1 recombinants able to inhibit its own replication as well as replication of wild-type virus.