Human cytomegalovirus (HCMV) genome manipulation has always been difficult. Recently, the introduction of full-length HCMV DNA into Escherichia coli as an artificial bacterial chromosome (BAC) clone has allowed reliable targeted mutagenesis. Here, we show the next generation of improvement in designing recombinant HCMV, which will also be applicable to other viral BAC clones. An inducible origin of replication linked with an antibiotic resistance marker was used as a cassette for targeted replacement of sequences within a HCMV BAC clone, TowneBAC. The origin of replication allowed for the induction of increased amounts BAC DNA that improved recovery, ease of use and transfections for mutant viruses. By specific deletion of UL147 and the recombinant GFP gene, we have shown that targeted deletion of a gene and selection for a recombinant genome are coupled with the ability to amplify the BAC clone DNA. These HCMV BAC clones were amplified approximately 10-fold. In the case of the removal of GFP from the clone TowneBAC shown in this study, the resulting BAC DNA preparation following amplification was used for successful primary cell transfection. Both parental and deletion BAC clone transfections gave similar levels of recombinant HCMV, and the GFP deletion virus replicated the same as the TowneBAC in a multi-step growth curve analysis.