In vitro labeling of DNA molecules using the Escherichia coli exonuclease III/DNA polymerase I enzyme pair has been examined as an alternative to existing methods of replacement synthesis labeling. It is shown that exonuclease III is able to act in a common restriction enzyme buffer [50 mM Tris (pH 8.0), 10 mM MgCl2, 50 mM NaCl] to produce a population of base-paired primer:template molecules which decrease uniformly in single-strand length with time. After heat inactivation of the exonuclease III and in the presence of radiolabeled deoxynucleotides the polymerase I reaction faithfully resynthesizes full-length molecules, asymmetrically labeled to high specific activity.