We studied the mechanism(s) of resistance to ciprofloxacin arising in a clinical strain of Pseudomonas aeruginosa and in a laboratory-derived isolate of that strain. Higher concentrations of ciprofloxacin were required to interfere with DNA synthesis in resistant isolates compared with the parent strain, a finding indicating a relative insensitivity of DNA gyrase to ciprofloxacin. Reduced uptake of ciprofloxacin was seen in one isolate and may have contributed to its ciprofloxacin resistance but was not associated with alterations in outer or cytoplasmic membrane proteins, a result suggesting that such changes are not required to decrease uptake of fluoroquinolones into cells. No evidence for plasmid-mediated resistance was found, and no ciprofloxacin-inactivating activity was detected in sonic extracts of resistant organisms. In these isolates, resistance to ciprofloxacin was likely the result of more than one mutation, because single-step mutations conferring such high-level resistance were not found in the parent strain.