Polynucleotide phosphorylase from cells of the cyanobacterium Synechococcus sp. has been purified 1400-fold by an improved procedure. The enzyme purified to homogeneity and lacking nuclease, phosphatase and protease contaminations reveals a single band of ADP polymerizing activity upon polyacrylamide gel electrophoresis under nondenaturing conditions which corresponds to a molecular mass of about 275 000. The enzyme migrates as a single polypeptide of Mr≈70 000 when subjected to gel electrophoresis in the presence of dodecyl sulfate indicating a composition of α4 for the native enzyme molecule. The isoelectric point of the purified enzyme as determined by isoelectric focusing was found to be at 4.2±0.1. Polynucleotide phosphorylase of Synechococcus is preferentially activated by Mg2+; Kcl has a significant stimulatory effect. © 1982, Walter de Gruyter. All rights reserved.