The use of live attenuated bacterial vaccine strains allows the targeted delivery of macromolecules to mammalian cells and tissues via the mucosal route. Depending on their specific virulence mechanisms and inherent metabolic preferences, bacteria invade certain cell types and body niches where they consequently deliver their cargo. Recently, the ability of attenuated strains of Salmonella, Shigella and Yersinia spp., as well as Listeria monocytogenes and invasive Escherichia coli, to deliver eukaryotic expression plasmids into mammalian cells in vitro and in vivo has been discovered. The great potential of bacteria-mediated transfer of plasmid DNA encoding vaccine antigens and/or therapeutic molecules was demonstrated in experimental animal models of infectious diseases, tumours and gene deficiencies. The exact mechanism of DNA transfer from the bacterial vector into the mammalian host is not yet completely known. The understanding of molecular events during bacterial DNA transfer, however, will further the development of bacterial vector systems with great promise for various clinical applications.