To test the suitability of simian virus 40 (SV40) DNA as a vector for inserting DNA segments into the chromosomes of mammalian cells, an EcoRI-A fragment of bacteriophage lambda DNA was covalently joined to a fragment of SV40 DNA and used to transform mouse cells in culture. Three independent, morphologically transformed clones were obtained that were positive for SV40 T-antigen by immunofluorescence staining. DNA from each transformant was examined by restriction enzyme analysis and found to contain both lambda and SV40 sequences. Co-migration of some fragments containing lambda and SV40 sequences following digestion of transformed cell DNA by each of four different restriction enzymes indicated that part of the retained lambda and SV40 DNA was linked in two of the three lines. In the third line, however, none of the restriction fragments had both lambda and SV40 sequences. Although the presence of non-integrated lambda DNA was not excluded, at least some of the lambda DNA appeared to be linked to host cell DNA. Results of digestion by EcoRI suggested that in some cases the transforming linear molecule had probably circularized prior to integration.