Intensive use of antimicrobial agents in health care settings not only leads to the selection of multiresistant nosocomial isolates of Staphylococcus aureus but may also promote endogenous, resistance-conferring mutations in bacterial genes that encode drug targets. We evaluated the spectrum of rifampin resistance-conferring mutations in cultures of methicillin-susceptible S. aureus (MSSA) or methicillin-resistant S. aureus (MRSA) strains exposed in vitro to sub-MICs of ciprofloxacin. Growth of ciprofloxacin-susceptible MRSA strain MRGR3 and ciprofloxacin-resistant MSSA strain RA1 (a NCTC 8325 derivative) in the presence of 1/2× or 1/4× MIC of ciprofloxacin led to higher frequencies of rifampin-resistant mutants on agar supplemented with rifampin (0.25 mg/liter) than under ciprofloxacin-free conditions. While rifampin-resistant mutants from ciprofloxacin-free cultures essentially showed single-amino-acid substitutions, a significant proportion of rifampin-resistant mutants from ciprofloxacin-exposed cultures displayed in-frame deletions or insertions in the rpoB gene at several positions of the rifampin resistance cluster I. In-frame deletions or insertions were also recorded in rpoB cluster I of rifampin-resistant mutants from ciprofloxacin-exposed cultures of mutS and mutL DNA repair mutants of ciprofloxacin-resistant S. aureus strain RA1. Frequencies of rifampin-resistant mutants grown under ciprofloxacin-free medium were higher for mutant strains RA1 mutS2 and RA1 mutL, but not RA1 recA, than for their parent RA1. In conclusion, ciprofloxacin-mediated DNA damage in S. aureus, as exemplified by the wide diversity of deletions or insertions in rpoB, suggests the occurrence of major, quinolone-mediated disturbances in DNA fork progression and replication repair. Besides promoting antibiotic resistance, accumulation of unrepaired DNA replication errors, including insertions and deletions, may also contribute to potentially lethal mutations.