DNA gyrase, an essential enzyme that regulates DNA topology in bacteria, is the target of fluoroquinolones. Three fluoroquinolone-resistant mutants derived from one strain of Clostridium perfringens had amino acid substitutions of glycine 81 to cysteine, aspartic acid 87 to tyrosine, or both, in alpha-helix-4 of gyrase A. The gyrase mutations affected the growth kinetics of mutants differently when the mutants were exposed to increasing concentrations of gatifloxacin and ciprofloxacin. Fluoroquinolone concentration-dependent effects observed during growth in the exponential and stationary phases depended on the presence of particular gyrA mutations. Introduction of a wild-type gyrA gene into the mutants enhanced their susceptibility to fluoroquinolones and decreased their growth rates proportional to increases in fluoroquinolone concentrations. Amino acid substitutions in alpha-helix-4 of gyrase A protected C. perfringens from fluoroquinolones, and a strain with two substitutions was the most resistant.