The activity of 4-quinolone antibacterials at the enzyme target level is based on the well known and reported observations that 4-quinolone antibacterials target the Gyr A subunit of the DNA gyrase holoenzyme, inhibiting supercoiling while facilitating the "cleavable complex". Such inhibition can be observed by running the in vitro DNA gyrase supercoiling inhibition assay or the "cleavable complex" DNA gyrase assay. Although potency of the gyrase inhibitor is dependent on many factors including permeability and pharmacokinetics, the inherent potency of a gyrase inhibitor lies in its activity against the target enzyme. We have examined the binding activity of novel flavones [Bioorganic & Med. Chem. Letters 3:225-230, 1993] to Escherichia coli DNA gyrase and have found differences in binding consistent with inhibition of DNA gyrase supercoiling and ability to facilitate the cleavable complex, but of different rank order. [3H]norfloxacin was used in vitro competition studies with test compounds, pBR322 and E. coli DNA gyrase. Binding affinity results indicate the rank order of greatest to weakest binding (ability to compete with [3H]norfloxacin) of test compounds: Levofloxacin = ciprofloxacin > ofloxacin > norfloxacin > flavone compounds (including ellagic acid, quercetin, and compounds 5a through 5n [Bioorganic & Med. Chem. Letters 3:225-230, 1993]). Such differences in binding ability of the 4-quinolones and flavones to the ternary complex of DNA.DNA gyrase.drug, as compared to the catalytic inhibition and "cleavable complex" data, suggests a more complex binding of flavones than the previously hypothesized models for 4-quinolone binding.