Fluoroquinolone MICs are increased through the acquisition of chromosomal mutations in the genes encoding gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE), increased levels of the multidrug efflux pump AcrAB, and the plasmid-borne genes aac(6′)-Ib-cr and the qnr variants in Escherichia coli. In the accompanying report, we found that ciprofloxacin, gatifloxacin, levofloxacin, and norfloxacin MICs for fluoroquinolone-resistant E. coli clinical isolates were very high and widely varied (L. Becnel Boyd, M. J. Maynard, S. K. Morgan-Linnell, L. B. Horton, R. Sucgang, R. J. Hamill, J. Rojo Jimenez, J. Versalovic, D. Steffen, and L. Zechiedrich, Antimicrob. Agents Chemother. 53:229-234, 2009). Here, we sequenced gyrA, gyrB, parC, and parE; screened for aac(6′)-Ib-cr and qnrA; and quantified AcrA levels in E. coli isolates for which patient sex, age, location, and site of infection were known. We found that (i) all fluoroquinolone-resistant isolates had gyrA mutations; (ii) ∼85% of gyrA mutants also had parC mutations; (iii) the ciprofloxacin and norfloxacin MICs for isolates harboring aac(6′)-Ib-cr (∼23%) were significantly higher, but the gatifloxacin and levofloxacin MICs were not; (iv) no isolate had qnrA; and (v) ∼33% of the fluoroquinolone-resistant isolates had increased AcrA levels. Increased AcrA correlated with nonsusceptibility to the fluoroquinolones but did not correlate with nonsusceptibility to any other antimicrobial agents reported from hospital antibiograms. Known mechanisms accounted for the fluoroquinolone MICs of 50 to 70% of the isolates; the remaining included isolates for which the MICs were up to 1,500-fold higher than expected. Thus, additional, unknown fluoroquinolone resistance mechanisms must be present in some clinical isolates.