We have developed a cosmid and plasmid system to generate mutant strains of murine cytomegalovirus (MCMV). The system is based on a series of seven overlapping cosmid clones that regenerate MCMV when cotransfected into mouse cells. The unaltered cosmids produce MCMV that is indistinguishable from wild-type MCMV based on restriction enzyme digest patterns of virus DNA and growth rates both in vitro and in vivo. Analysis of viral DNA from plaque-purified recombinant isolates taken from in vitro and in vivo stocks indicated that regeneration did not introduce novel mutations in the recombinant viral genomes. Isolation of specific genes and subsequent generation of specific mutant MCMVs was accomplished by replacement of cosmids with overlapping plasmid subclones. A new vector, PmeSUB, featuring a multiple cloning site and a stringent origin of replication, was constructed to make large subclones for use with smaller subclones containing the gene of interest. The utility of this system was demonstrated by the generation of two different mutant MCMVs from different combinations of overlapping plasmid subclones of one cosmid. The advantages of this system are that (i) target genes are maintained as small clones making them amenable to standard in vitro mutagenesis manipulations and that (ii) no reporter or selection genes are necessary to identify mutants.