T4 RNA ligase joins a 3'-hydroxyl-terminated acceptor oligoribonucleotide to a 5'-phosphate-terminated donor oligoribonucleotide. An analogous reaction with single-strand DNA oligonucleotides would be useful for the synthesis of defined sequences of DNA because it would eliminate the need to synthesize complementary sequences to form the duplex substrates required by DNA ligase. We have studied the model reaction dA(pdA)5 + [5'-32P] (pdT)4pdCp leads to dA(pdA)5 [3' leads to 5'-32P]pdT(pdT)3pdCp and have obtained 40-60% yields at equimolar concentrations (100 microM to 1 mM) of the two substrates. Higher yields have been obtained when acceptor concentrations in excess of those of the donor are used. The use of a 5'-hydroxyl, 3'-hydroxyl terminated acceptor and a 5'-phosphate, 3'-phosphate terminated donor limits the reaction to a unique product. The 3'-phosphate-terminated donor was prepared by using RNA ligase to add a single deoxyribonucleoside 3',5'-bisphosphate donor to an oligo(deoxyribonucleotide) acceptor [Hinton, D.M., Baez, J.A., & Gumport, R.I. (1978) Biochemistry 17, 5091]. The DNA oligomer joining reaction requires low concentrations of ATP and an ATP regenerating system, Mn2+, high levels of nuclease-free RNA ligase (30 microM), and incubation for several days at 17 degrees C. The product of the reaction was characterized by its resistance to alkaline phosphatase, degradation by micrococcal nuclease to the expected product [3'-32P]dAMP, and mobility during high-pressure liquid chromatography on RPC-5. The joining of several other deoxyoligomers was also demonstrated. We anticipate that this reaction of RNA ligase will contribute to its usefulness as a reagent for the synthesis of DNA of defined sequence.