Treatment of Ltk-, mouse L cells deficient in thymidine kinase (tk), with Barn I restriction endonuclease cleaved DNA from herpes simplex virus-l (HSV-1) produced tk+ clones with a frequency of 1O-6/2 pg of HSV-1 DNA. Untreated cells or cells treated with Eco RI restriction endonuclease fragments produced no tk+ clones under the same conditions. The thymidine kinase activities of four independently derived clones were characterized by biochemical and serological techniques. By these criteria, the tk activities were found to be identical to HSV-1 tk and different from host wildtype tk. The tk+ phenotype was stable over several hundred cell generations, although the rate of reversion to the tk-phenotype, as judged by cloning efficiency in the presence of bromodeoxyuridine, was high (l-5 x 10m3). HSV-1 DNA Barn restriction fragments were separated by gel electrophoresis, and virtually all activity, as assayed by transfection, was found to reside in a 3.4 kb fragment. Transformation efficiency with the isolated fragment is 20 fold higher per gene equivalent than with the unfractionated total Barn digest. These results prove the usefulness of transfection assays as a means for the bioassay and isolation of restriction fragments carrying specific genetic information. Cells expressing HSV-1 tk may also provide a useful model system for the detailed analysis of eucaryotic and viral gene regulation.