An RNA ligase has been purified from HeLa cells, which catalyzes the intra- and intermolecular ligation of linear RNA substrates possessing 5'-hydroxyl and 2',3'-cyclic phosphate termini in the presence of ATP or dATP. In this reaction, the 2',3'-cyclic phosphate is incorporated into a 3'-5'-phosphodiester bond, in agreement with the findings of Filipowicz et al. [Filipowicz, W., Konarska, M., Gross, H. J. & Shatkin, A. J. (1983) Nucleic Acids Res. 11, 1405-1418]. The activity of the purified enzyme is dependent on the addition of ATP or dATP, a divalent cation (Mg2+), and 5'-hydroxyl, 2',3'-cyclic phosphate-terminated RNA substrates. No ligation occurs with the substrates OH(Up)10G(3')p or OH(Up)10G(2')p or with 5'-phosphate, 2',3'-cyclic phosphate-terminated oligoribonucleotides.