Rose Bengal is a potent inhibitor of the DNA-dependent RNA polymerase of Escherichia cali and photo-oxidation is not necessary for this inhibition. Virtually no inhibition occurs at 10–7 M Rose Bengal, while complete inhibition is reached at about 10–5 M. The concentration of Rose Bengal required to inhibit 50% of RNA polymerase activity is 1.4 X 10–6 M and is independent of the DNA or synthetic polynucleotides used as template. Kinetic analysis reveals a noncompetitive inhibition of Rose Bengal with respect to nucleoside triphosphates. This suggest that the inhibitor does not affect the combination of the substrate with the enzyme. Similarly, Rose Bengal (≤10–5 M) does not alter the binding of RNA polymerase to T7 DNA as measured by a nitrocellulose filter assay. At lower concentrations (10–6 M), Rose Bengal selectively inhibits RNA chain elongation compared to initiation (This was demonstrated by the differential effects of Rose Bengal on [3H]UMP vs. [γ-32P]ATP incorporation into RNA chains.), resulting in the formation of smaller RNA products. Rose Bengal also exhibits a selective inhibition of the DNA-dependent [32P]PPi-exchange reaction catalyzed by RNA polymerase. With d(A-T) copolymer, the exchange of [32P]PPi with UTP and ATP was inhibited by 10–6 M Rose Bengal, while this concentration had no effect on the [32P]PPi exchange when ATP was replaced by AMP, ADP, or UpA. It is suggested that Rose Bengal might either inhibit the formation of phosphodiester bonds or block the translocation of enzyme along the template. At higher concentrations (10–5 M), Rose Bengal also inhibits chain initiation and contributes to the premature release of nascent RNA chains from the enzyme-DNA complex. Furthermore, evidence is presented that Rose Bengal binds reversibly to RNA polymerase but does not bind to DNA.