T4 RNA ligase catalyzes the addition of a single deoxyribonucleoside 3',5'-bisphosphate to the 3'-hydroxyl of oligodeoxyribonucleotides (Hinton et al. (1978) Biochemistry 17, 5091). We have determined improved conditions for this reaction which give yields equal to or greater than 85% when any of five common deoxyribonucleoside bisphosphate (pdAp, pdCp, pdGp, pdTp, or pdUp) are added to dA(PDA)4. A low ATP concentration, which is constantly maintained by a regeneration system composed of phosphocreatine, creatine kinase, and myokinase, contributes to the attainment of high yields. The addition of RNase A and spermine also enhances the rates and yields of the reactions. These conditions facilitate the use of RNA ligase as a reagent for the stepwise synthesis of DNA of defined sequence.