Abstract RNA ligase has been extensively purified by a new procedure in high yield from T4-infected Escherichia coli . The enzyme consists of a single polypeptide chain of molecular weight 47,000. It catalyzes the formation of a phosphodiester bond between a 5′-PO 4 -terminated oligonucleotide and a 3′-OH terminated oligonucleotide. The purified enzyme catalyzes both the intramolecular formation of single-stranded circles with longer oligonucleotides of the type pAp(Ap) n A −OH , where n is about 15 or greater and the intermolecular joining of pAp(Ap) 3 A OH (where the 5′-PO 4 -terminated oligonucleotide is short enough to prevent apposition of its 3′ and 5′ ends) to UpUpU OH when high concentrations of the 3′-OH-terminated acceptor oligonucleotide are present. Preparations of RNA ligase at all stages of purification show an unusual dependence of specific activity of the enzyme on the concentration of enzyme present in the assay. However, when care is taken to determine meaningful specific activities at each step, the ligase is found to be very stable during chromatography on various ion-exchange columns and may be purified by conventional techniques.