Spontaneous quinolone-resistant mutants of MP050, a quinolone-susceptible clinical strain of Serratia marcescens, were isolated on nutrient agar containing 0.5 microgram of ciprofloxacin per ml. One mutant, designated MP051, was selected for further study. Quinolone MICs for MP051 were 4- to 16-fold higher than those for MP050; nonquinolone MICs were unchanged. The DNA gyrase isolated from MP051 was 24-fold less sensitive to inhibition of supercoiling by ciprofloxacin than the DNA gyrase isolated from MP050 was. Inhibition studies on reconstituted combinations of heterologous gyrase subunits showed that the decreased inhibition was dependent on the A subunit of DNA gyrase from MP051. Further evidence that this decreased inhibition was due to a gyrA mutation was provided by analysis of Escherichia coli gyrA gene expression in S. marcescens heterodiploids containing pNJR3-2, a broad-host-range gyrA gene probe. Quinolone susceptibilities of MP051 heterodiploids containing the wild-type E. coli gyrA gene decreased to those of MP050, while quinolone susceptibilities of MP050 containing the same plasmid were unchanged. These results indicate that spontaneous quinolone resistance in MP051 was due to a mutation in gyrA.