The mechanism of action of the quinolone analogs ofloxacin and S-25930, which are unusual because of the presence of a third ring with an asymmetric carbon, was studied. Drug-resistant strains of Escherichia coli were selected by serial passage in the presence of ofloxacin, and a mutation was mapped near the gyrA gene of DNA gyrase. DNA gyrase containing the A subunit purified from this strain as compared with the isogenic wild-type strain exhibited increased resistance to ofloxacin, proving that the mutation was located in the gyrA gene. For S-25930, the S stereoisomer was more potent than the R isomer in inhibiting wild-type E. coli and DNA gyrase containing an A subunit isolated from this strain. Both isomers had decreased potency against the isogenic ofloxacin-resistant (gyrA) strain and its purified enzyme, but the S isomer remained more potent than the R isomer. These studies, using a combined genetic and biochemical approach, demonstrate (i) that DNA gyrase is a target of the tricyclics ofloxacin and S-25930, (ii) that serial exposure to ofloxacin can select resistance to tricyclic quinolone agents by mutation in the gyrA gene, and (iii) that the more potent antibacterial activity of S relative to R S-25930 correlates with increased activity against DNA gyrase for both wild-type and ofloxacin-resistant (gyrA) isogenic strains.