Abstract A shuttle-vector system is described for the study of mutational specificity in mammalian cells. Using a plasmid (pGKTK) carrying the E. coli galactokinase gene ( gk) and the herpes simplex virus thymidine kinase gene ( tk), we demonstrate the introduction of a foreign gene into the chromosome of a mammalian cell (TK − mouse fibroblasts) and its efficient rescue back into E. coli. The system makes use of two genes, each of which can expressed in both E. coli and mammalian cells, thereby permitting one marker to be the mutational target and the other to maintain stable integration in the host. In addition, expression of both genes in bacteria makes it possible to deletion map mutants to facilitate their sequencing. In the case of a putative single-copy transformat (T8), about half of the rescued plasmid are identical in size and restirction pattern to the original plasmid. Each of these expressed the tk gene, indicating the fidelity of the rescue system.