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Enterococci isolated from Portuguese dairy products (milk and cheese) and clinical settings (hospitals and veterinary clinics), together with reference strains from the genus Enterococcus, were screened for low- and high-level gentamicin resistance using the standard disc diffusion method (10 and 120 µg gentamicin discs). MICs were also determined using both the macrodilution method and the Etest. Four genes [aac(6′)-Ie-aph(2″)-Ia, aph(2″)-Ib, aph(2″)-Ic and aph(2″)-Id] responsible for high- and mid-level gentamicin resistance were sought using PCR. Although enterococci generally are regarded as being intrinsically resistant to low levels of gentamicin, results revealed that many dairy enterococci (around 30% of the isolates used) are not intrinsically resistant to gentamicin, showing MICs of £4 mg/L. High-level gentamicin resistance was not detected in any of the dairy isolates studied, except for aph(2″)-Ib, which was found in one. Therefore, gentamicin resistance should be monitored in dairy enterococci, although it does not seem to be a problem at present. In contrast, all clinical isolates studied were, as expected, intrinsically resistant to low levels of gentamicin, presenting MICs > 8 mg/L. Fifteen percent of these clinical isolates showed high-level gentamicin resistance (MICs > 512 mg/L), with the bifunctional gene aac(6′)-aph(2″) being detected in four of them. However, discs with gentamicin 120 µg failed to detect some isolates with high-level gentamicin resistance.