The oligonucleotides pppA2'p5'A2'p5'A and related oligomers (2-5A) are synthesized by an enzyme that is widely distributed in a variety of cells, the activity of which varies with interferon treatment, growth and hormone status. Because significant amounts of 2-5A have recently been detected in interferon-treated cells, it has been suggested that the oligonucleotides may be involved in interferon action and in the control of cell metabolism. In both intact cells and cell-free systems 2-5A has been shown to activate a ribonuclease. We report here investigations of the sequence specificities of the 2-5A-dependent ribonucleases in extracts of rabbit reticulocytes, mouse ascites tumour cells and human lymphoblastoid cells in conditions of partial digestion using terminally labelled RNA substrates. The enzymes cleaved on the 3'-side of UN sequences to yield UpNp terminated products. Cleavage was observed predominantly at UA and UU sequences.