Bis(1 -anilino-8-naphthalenesulfonate) [bis(Ans)] is a dimer of l-anilino-8-naphthalenesulfonate with a C-C linkage at the 4,4ʹ positions of the naphthalene rings. More than 90% of RNA polymerase activity is inhibited by 10-5 M bis(Ans), while no inhibition is observed with Ans at the same concentration. The concentration of bis(Ans) required for 50% inhibition is about 4 X 10-6 M using various DNA templates. The inhibition can be partially prevented by preincubation of the enzyme with DNA and/or nucleoside triphosphates. At 10-5 M, bis(Ans) has no effect on the binding of RNA polymerase to DNA as measured by retention of the enzyme-DNA complex on a nitrocellulose filter. However, little DNA retention was detected with 10-4 M bis(Ans). Kinetic studies and the differential effects of bis(Ans) on 3H-labeled vs. γ-32P-labeled nucleotide incorporations into the RNA product indicate that bis(Ans) at concentrations of ≤10-5 M selectively inhibits RNA chain initiation. Using T7 DNA as tem late, we have found that bis(Ans) inhibits the synthesis of the dinucleotide pppGpC catalyzed by RNA polymerase. Bis(Ans) binds to enzyme but not DNA as demonstrated by gel filtration and fluorescence spectroscopy. The binding of bis(Ans) to RNA polymerase has been studied by a fluorimetric titration technique. The binding isotherms show multiple binding sites of the dye, which can be divided into two distinct classes: a class of 16-18 strong binding sites with apparent Kd = 2X 10-6 M and a class of 34-36 weak binding sites with apparent Kd = 2 X 10-5 M. Glycerol-gradient centrifugation analysis has revealed that RNA polymerase dimerizes in the presence of 1 X 10-4 M bis(Ans) but remains as a monomer at 1 X 10-5 M bis(Ans) in the high-salt (0.5 M KC1) buffer. Both the fluorescence and sedimentation results indicate that binding of bis(Ans) to the strong sites on enzyme inhibits RNA chain initiation, whereas binding to the weak sites induces dimerization of the enzyme concomitant with loss of its ability to bind DNA template. Further fluorescence studies show that aromatic amino acid residues are involved in the binding of dye.