Inhibitory activity of test compounds against Escherichia coli DNA gyrase in a "cleavable complex" assay, readily observed in vitro at the enzyme level by the artificial addition of a denaturing agent, is found to be an excellent indicator of 4-quinolone inhibition of DNA gyrase, and as accurate a predictor of target enzyme inhibitory activity as the measurement of the inhibition of DNA gyrase supercoiling. This study was designed to examine the specificity of DNA gyrase inhibitors of various chemical classes in these two DNA gyrase assays, and define the use of these two assays in understanding the nature of inhibition by experimental compounds. Supercoiling inhibition was detected by determination of the 50% inhibition level, and cleavable complex inhibition measured by the determination of the drug concentration at which 50% of the maximal (of control) formation of linear, cleaved DNA was obtained. Results indicate that these two assays can serve several different functions in microbiological research, among them: (1) quantitation of enzyme inhibitory activity at the target level; and (2) distinguishing between nonspecific inhibition or artifactual inhibition of DNA gyrase and true, mechanism-based inhibition of the catalytic activity of DNA gyrase.