Interaction of Rose Bengal with RNA polymerase of Escherichia coli has been studied by absorption and fluorescence spectroscopy. Upon binding to RNA polymerase, the absorption maximum of Rose Bengal shifted from 546 to 561 nm, while the fluorescence emission maximum shifted from 567 to 575 nm. The protein-induced enhancement in fluorescence intensity of Rose Bengal (the quantum yield changed from 0.013 to 0.12), however, was much greater than the increase in absorbance (an 18% increase in extinction coefficient). The stoichiometry and dissociation constants of the enzyme-inhibitor complex were determined by difference-spectrophotometric and fluorimetric titrations. In both cases, a dissociation constant of 6 X 10–7 M was obtained. This value is in good agreement with the Ki value (7 X 10–7 M) calculated from kinetic studies. The number of Rose Bengal binding sites estimated on RNA polymerase is 1.3/enzyme molecule (mol wt 500,000). In order to gain insight into the nature of the Rose Bengal binding site, the effect of solvent polarity on the fluorescence properties of Rose Bengal has been investigated. It was concluded that the environment of the Rose Bengal binding site on RNA polymerase is highly nonpolar and perhaps consists of a hydrophobic pocket. Furthermore, the fluorescence of tryptophan residues of RNA polymerase was quenched on addition of Rose Bengal. Excitation spectrum of the Rose Bengal-enzyme complex indicates that this is due to energy transfer from tryptophan residues to the bound dye. The efficiency of transfer is markedly decreased by addition of DNA and NTP.1 This could be explained by template and substrate-induced conformational changes of RNA polymerase. Although the binding of Rose Bengal markedly alters the catalytic properties of RNA polymerase, all the experimental results are consistent with a picture in which the Rose Bengal binding site is spatially distinct from the template or substrate binding sites on the enzyme.