Fluoroquinolones are poorly active against enterococci. Recently, plasmid-borne resistance to fluoroquinolones due to the qnr gene was reported in members of the Enterobacteriaceae family. The gene encodes a pentapeptide repeat protein that protects DNA gyrase from inhibition by fluoroquinolones. We have identified in the genome of Enterococcus faecalis V583 a qnr-like gene, named E. faecalis qnr (qnr(E. faecalis)), encoding a putative pentapeptide repeat protein that shares 25% identity with Qnr. To assess its potential role in the intrinsic resistance of E. faecalis to fluoroquinolones, qnr(E. faecalis) was inactivated in E. faecalis JH2-2 by insertion of the thermosensitive vector pG1KT. This strain was then complemented with qnr(E. faecalis) cloned in the multicopy plasmid pORI23. The effects of its overexpression were also studied. Inactivation of the qnr(E. faecalis) gene resulted in twofold decreases in the MICs of ofloxacin and ciprofloxacin. When the gene was complemented or overexpressed, MICs of fluoroquinolones increased four- to nine-fold, leading to MICs of ofloxacin and ciprofloxacin equal to 32 mug/ml and 8 mug/ml, respectively. The E. faecalis Qnr (Qnr(E. faecalis)) protein was produced and purified. Qnr(E. faecalis) protein protected Escherichia coli DNA gyrase from inhibition by ofloxacin. The qnr(E. faecalis) gene was then introduced into E. coli DH10B, Staphylococcus aureus RN4220, and Lactococcus lactis IL-1419 to study its heterologous expression. MICs of the various fluoroquinolones tested increased 4- to 16-fold, showing that Qnr(E. faecalis) conferred resistance to fluoroquinolones in various bacterial backgrounds. Overexpression of qnr(E. faecalis) in enterococci or mobilization of the gene to other bacterial species may be anticipated as a possible new mechanism for fluoroquinolone resistance.