We describe SV40-based Escherichia coli shuttle vectors which can be packaged as pseudovirions without excision of plasmid sequences and which can be rescued in bacteria. These vectors replicate and are transmitted as virus in monkey COS cells without requiring a helper virus. Extrachromosomal vector DNA isolated from infected cells can be rescued in E. coli, so that DNA alterations can be easily screened. Indeed, some of the constructions give rise to very stable plasmids with no detectable rearrangements after multiple lytic cycles in COS cells. The spontaneous mutation frequency measured in bacteria, on the lacO target, is smaller than those usually found with shuttle vectors. We also constructed an expression vector derived from one of our infectious viruses by inserting the chloramphenicol acetyl transferase gene, expressed from the SV40 early promoter, which is efficiently transduced to cells by infection. In this system, the shuttle virus combines the convenience of plasmid rescue and analysis in bacteria, with the advantages of infectious virus.