We have constructed a shuttle vector plasmid for studying mutagenesis in mammalian cells. The plasmid replicates in cell lines permissive for SV40 virus as well as in the bacterium Escherichia coli and carries a bacterial suppressor tRNA gene (supF) that can serve as a mutagenesis marker. The plasmid replicates as efficiently as SV40 virus in African Green Monkey kidney CV1 cells, indicating that all traces of the inhibitory sequences normally found in pBR322 and its derivatives have been removed. The design of the plasmid and the small size of the mutagenesis target gene decrease the probability of recovering spontaneous deletion mutations that have been shown to occur at high frequency during passage in mammalian cells. The frequency of spontaneous-mutant plasmids recovered after passage in CV1 cells is substantially lower than with other vectors described previously. When the plasmid DNA is treated with UV radiation before passage in CV1 cells, mutants are observed at a frequency about 20-fold above the spontaneous background.