The Varicella-Zoster Virus (VZV) genome contains both cis-acting and trans-acting elements, which are important in viral DNA replication. The cis-acting elements consist of two copies of oriS, and the trans-acting elements are those genes whose products are required for virus DNA replication. It has been shown that each of the seven genes required for ori-dependent DNA synthesis of Herpes Simplex Virus type-I (HSV) has a homologue in the VZV genome. However these seven genes of VZV never supported replication of the plasmid containing VZV oriS, in the same assay. On the other hand, several laboratories have shown that co-transfected cosmid DNAs containing the overlapping VZV fragments can supply all of the necessary functions for the VZV virion development. In the present study we found that the four overlapping cosmids are as efficient as virion in supporting the in vitro replication of co-transfected plasmids containing oriS. On the basis of these results, subcloning of each of the cosmids used in this system should allow one to identify all of the VZV genes required for DNA replication in cultured cells.