We describe a new, rapid, and convenient procedure for purifying 2',5'-oligoadenylate synthetases, employing precipitation with ammonium sulfate, fractionation by gel filtration, rapid binding to poly(I) X poly(C) cellulose, and elution with 0.35 M KCl. Unlike previously published methods, the procedure does not require sedimentation of the enzyme at 200,000 X g. Therefore, it is more general and more likely to succeed with synthetases extracted from a variety of cells or tissues, or from different subcellular fractions. We have purified the enzymes from two sources to apparent homogeneity, about 2500-fold from the cytoplasm of HeLa cells in 40% yield and more than 400,000-fold from the cytoplasm of rabbit reticulocytes in 25% yield. The specific activity of the HeLa enzyme is about 4 times higher than reported previously. The physical and functional properties of the pure enzymes are very similar to those reported by others for preparations of 2',5'-oligoadenylate synthetase from rabbit reticulocytes, mouse L cells, and human HeLa cells. A new affinity matrix was prepared by linking periodate-oxidized poly(I) X poly(C) to a hydrazide derivative of finely divided cellulose. Poly(I) X poly(C) cellulose binds about twice as much synthetase as the corresponding amount of poly(I) X poly(C) paper and activates the bound enzyme about three times better.