We provide evidence of direct transfer of functional DNA from bacteria to mammalian cells. An Escherichia coli K12 diaminopimelate auxotroph made invasive by cloning the invasin gene from Yersinia pseudotuberculosis transfers DNA after simple co-incubation, into a variety of mammalian cell lines. Transfer efficiency was enhanced in some cells by coexpression of the gene for listeriolysin from Listeria monocytogenes. Expression of the acquired genes occurs in both dividing and quiescent cells. The only requirement for bacteria to transfer genetic material into nonprofessional phagocytic cells and macrophages is the ability to invade the host cell.