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 microg 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 < or = 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 microg failed to detect some isolates with high-level gentamicin resistance.