A strategy employing T4 DNA polymerase replacement synthesis is described whereby only the insert portion of recombinant plasmids are radioisotopically labeled. Prior purification of the inserted DNA is not required. The recombinant plasmid is first digested with one or more restriction endonucleases selected to cleave the vector segment into fragments at least 30% shorter than the insert DNA segment. This mixture of fragments is then digested by the T4 DNA polymerase-associated 3' exonuclease in the absence of deoxynucleoside triphosphates (dNTPs) for a length of time which allows complete degradation of all fragments shorter than the insert. The remaining insert DNA, which is now partially single-stranded, is then resynthesized by addition of dNTPs, one or more of which is labeled. The resulting DNA is full length, double-stranded, and unnicked. The strategy is widely applicable, and reliably and reproducibly yields DNA of high specific activity. We have used this method to label more than 15 cloned inserts ranging in size from 3.2 to 25 kilobases.