Cibacron blue F3GA is a potent inhibitor of the Azotobacter vinelandii DNA-directed RNA polymerase. Addition of 8 micrometer Cibacron blue F3GA prior to initiation results in a greater than 90% inhibition of the poly[d(A-T]-directed synthesis of poly[r(A-U)] while addition of the dye during the course of the reaction is without effect on chain elongation. Binding of RNA polymerase to [3H]poly[d(A-T)] is inhibited by only 15% in the presence of 8 micrometer Cibacron blue F3GA. Inhibition by Cibacron blue F3GA is noncompetitive with regard to ATP, UTP, or template. The poly[d(A-T)]-directed pyrophosphate exchange reaction is relatively resistant to inhibition by Cibacron blue F3GA. Rifampicin added to a similar reaction (in the presence of absence of Cibacron blue F3GA) results in 95% inhibition of the exchange reaction. The interaction of the RNA polymerase core enzyme with Cibacron blue F3GA is shown by the formation of a difference spectrum with a positive maximum at 675 nm which is not affected by the presence of a high concentration (4 micrometer) of rafampicin. The data indicate that Cibacron blue F3GA acts by binding to RNA polymerase and inhibits a step between the synthesis of the initial phosphodiester bond and formation of a stable ternary elongation complex.