The effect of salt concentration on RNA synthesis by DNA-dependent RNA-polymerase from Escherichia coli has been investigated. For a double stranded template the following influences are observed: (1) Free enzyme and enzyme bound to DNA, but not synthesizing RNA, are inhibited above an ionic strength of about 0.15. (2) The rate of RNA-synthesis, by those enzyme molecules which have started RNA chains, is stimulated by increasing the salt concentration up to an optimum. Magnesium salts cause a larger stimulation than monovalent cations. However, the optimal effect always occurs at an ionic strength of about 0.36. (3) This stimulatory effect is distinguished from the cofactor function which requires only low concentrations of divalent cations (Mg2+, Co2+, Mn2+). (4) RNA synthesis stops at low ionic strength (< 0.2) due to a first order inactivation of the synthesizing enzyme. A t high ionic strength synthesis continues over many hours a t a slowly decreasing rate. Raising the ionic strength after RNA synthesis has stopped at low ionic strength causes an immediate resumption of synthesis. Mg2+ and spermidine are most effective in this action. However, Co2+ and Mn2+ are unable to reactivate the enzyme once it has stopped.With single stranded DNA as template neither a first order inactivation of synthesizing enzyme a t low ionic strength nor a stimulation of RNA synthesis by monovalent> salts is observed. The effect of divalent cations on enzyme activity can be due to their cofactor function.The DNA-dependent RNA-polymerase from Escherichia coli is strongly influenced by changes in the ionic strength. At low ionic strength (between 0.02 and 0.12) it has been shown to exist in a form sedimenting a t 24s [l-51; a t high ionic strength the enzyme reversibly dissociates into subunits of 13s [2,4-61. At low salt concentration the polymerase forms a complex with DNA [1,7--12j which by raising the ionic strength is dissociated into free DNA and the i3S form of the enzyme [Z, 13-15].The polymerization process itself has been reported to be inhibited only after some time of synthesis at high ionic strength [5,13]. However, the effect of ionic strength on this phase of the reaction has not been extensively investigated.This communication presents a detailed analysis of the effect of ionic strength on the kinetics of RNAsynthesis with double and single-stranded DNA. It is shown that increasing the ionic strength causes Non-Standard Abbreviations. PPO, 2,5 diphenyloxazol; POPOP, 1,4-bis-2-(4 methyl4 phenoxazoly1)-bcnzene; TMA, buffer, containing: 0.01 M Tris acetate, 0.022 M NH,CI, 0.01 M magnesium acetate, 1 mM 2-mercaptoethanol, pH 7.3; TCA, trichloroacetic acid.Enzymes. DNA-dependent RNA-polymerase or nucleosidetripliosphate : RNA nucleotidyltransferase (EC 2.7.7.6) ; Ribonuclease I (EC 2.7.7.17); Pyruvate kinase (EC 2.7.1.40). a striking stimulation of both the rate and extent of RNA synthesis. This eliminates the typical plateau in the kinetics [l6-211 and allows RNA-synthesis t o continue over many hours. Moreover, synthes...