The polynucleotide ligase isolated from T4-infected Escherichia coli was previously shown to bring about repair of breaks in the single strands of bihelical DNA. The present work shows that the enzyme can also catalyze the joining of DNA duplexes at their base-paired ends. This novel reaction occurs when the deoxynucleoside at a 5′-end carries a phosphate group and the complementary deoxynucleoside opposite to it carries a 3′-hydroxyl group. The consequence is the lengthening of the original duplex to form dimers or oligomers depending upon whether one or both ends are base-paired.