OBJECTIVE The major mechanism of fluoroquinolone (FQ) resistance in Pseudomonas aeruginosa (PSA) is modification of target proteins in DNA gyrase and topoisomerase IV, most commonly the gyrA and parC subunits. The objective of this study was to determine risk factors for PSA with and without gyrA or parC mutations. METHODS Case-case-control study METHODS Two adult academic acute-care hospitals METHODS Case 1 study participants had a PSA isolate on hospital day 3 or later with any gyrA or parC mutation; case 2 study participants had a PSA isolate on hospital day 3 or later without these mutations. Controls were a random sample of all inpatients with a stay of 3 days or more. METHODS Each case group was compared to the control group in separate multivariate models on the basis of demographics and inpatient antibiotic exposure, and risk factors were qualitatively compared. RESULTS Of 298 PSA isolates, 172 (57.7%) had at least 1 mutation. Exposure to vancomycin and other agents with extended Gram-positive activity was a risk factor for both cases (case 1 odds ratio [OR], 1.09; 95% confidence interval [CI], 1.04-1.13; OR, 1.14; 95% CI, 1.03-1.26; case 2 OR, 1.09; 95% CI, 1.03-1.14; OR, 1.13; 95% CI, 1.01-1.25, respectively). CONCLUSIONS Exposure to agents with extended Gram-positive activity is a risk factor for isolation of PSA overall but not for gyrA/parC mutations. FQ exposure is not associated with isolation of PSA with mutations.