Abstract The dependence of Micrococcus lysodeikticus RNA polymerase on divalent cations was studied in vitro with the use of synthetic polynucleotides as templates. It was found that the various combinations of manganese or magnesium cofactors with polyuridylic, polycytidylic, and polyadenylic acid templates had characteristic activity profiles for the chain initiation and elongation steps. To distinguish between the initiation of chain synthesis and its elongation, appropriate pentanucleotides were added to bypass the rate-limiting chain initiation reaction. In the copying of polycytidylic acid, the divalent cation requirement was independent of substrate concentration. The synthesis of polyadenylate and polyuridylate, however, evinced sharp metal ion optima at levels approximately equimolar with the total ligand concentration (i.e. nucleoside triphosphate plus other chelating species). Whereas manganese stimulated chain initiation and elongation in all functioning polymer systems, magnesium ion exhibited great selectivity in the initiation step. Thus, Mg2+ failed to promote the initiation of polyadenylic and polyuridylic acid synthesis, but still stimulated the elongation of the corresponding exogenous pentanucleotide chains. Polyguanylic acid was the only homopolymer the synthesis of which was initiated by Mg2+. The relevance of this model system to the problem of the initiation of RNA synthesis in vivo is discussed.