Abstract After mixed infection of Escherichia coli strain BB with 32P-labeled- and bromouracil density-labeled T4 am EB6 (a T4 mutant defective in the DNA polymerase gene), two kinds of hybrid molecules were isolated. One type contained the 32P- and bromouracil-labeled components linked only by hydrogen bonds (joint molecules); the second type contained the labeled components in covalent linkage (recombinant molecules). Infection with 32P-labeled- and bromouracillabeled T4 am EB6-605 (a T4 mutant defective in both the DNA polymerase and polynucleotide ligase genes) led to the formation of only joint DNA molecules. These results indicate that a functional T4-induced ligase is required for the formation of recombinant DNA molecules in vivo. Conversion of joint to recombinant molecules was achieved in vitro and required ligase, DNA polymerase and the four deoxynucleoside triphosphates. This finding suggests that joint molecules are double-stranded structures in which single-strand gaps separate the polynucleotide segments derived from the parental DNA molecules.