The interaction of nucleoside triphosphates with ribonucleic acid polymerase of Escherichia coli has been studied by the technique of equilibrium dialysis. In the absence of divalent metal, purine nucleotides bind to a single site on the enzyme (mol wt 370,000). Dissociation constants for guanosine triphosphate of 0.15 mM and for adenosine triphosphate of 0.17 mM were observed. Rifamycin inhibited completely the binding of purine nucleoside triphosphate in the absence of divalent metal. No binding of pyrimidine nucleoside triphosphates at concentrations up to 0.2 mM was observed in the absence of divalent metal. With 10 mM MgCl2, two binding sites for purine nucleoside triphosphates were observed. The weak site with a Ks of 0.15 mM was abolished by rifamycin leaving a single strong binding site with a Ks of 0.015 mM. A single weak binding site for pyrimidine nucleoside triphosphates was observed with a Ks for cytidine triphosphate of 0.23 mM and for uridine triphosphate of 0.37 mM. This binding was not sensitive to rifamycin. There was no enhancement of pyrimidine nucleoside triphosphate binding by guanosine triphosphate, or Mn, or at a lower pH. Some decrease in the Ks for uridine triphosphate was observed with transfer ribonucleic acid, and for cytidine triphosphate with polyadenylic acid. Uridine monophosphate was not bound. No evidence of phosphodiester bond formation was observed.