OBJECTIVE To investigate the nature of aminoglycoside resistance of clinically important multidrug-resistant (MDR) Acinetobacter baumannii strains from the Czech Republic. MATERIAL AND METHODS Seventy MDR hospital strains from 1991-2001 classified into pan-European clones I (n = 41) and II (n = 21) and a group of other MDR strains (n = 8) were studied. Fifteen other strains wild-type susceptible to aminoglycosides were used as controls. Susceptibility to aminoglycosides was determined by the disk diffusion and agar dilution tests. The presence of six genes encoding different aminoglycoside-modifying enzymes (AME) and of the adeB gene encoding a component of the AdeABC efflux pump was detected using PCR. RESULTS MDR strains showed the following pattern of resistance to aminoglycosides: kanamycin (93 %; inhibition zone diameters < or =13 mm), gentamicin (87 %; MICs > or =16 mg/l), amikacin (47 %; MICs > or =64 mg/l), tobramycin (31 %; MICs > or = 16 mg/l) and netilmicin (17 %; MICs ? 32 mg/l). Forty-six (66 %) MDR strains had netilmicin MICs of 8-16 mg/l in contrast to 0.5-1.0 mg/l found for control susceptible strains. The presence of at least one of the following AME genes was detected in 66 (94 %) MDR strains: aphA1 (n = 56), aacC1 (n = 52), aphA6 (n = 39) and aadB (n = 20). The presence of these genes and phenotypes of resistance to kanamycin, gentamicin, tobramycin and amikacin were in good agreement. The genes encoding netilmicin-modifying enzymes (aacC2, aacA4) were not detected in any strain. Fifty-six (80 %) MDR strains comprised two to four different AME genes in eight combinations. Clones I and II shared all of the genes studied (with the exception of aadB not detected in clone I). The gene adeB was found in all MDR strains and eight of 15 control susceptible strains. CONCLUSION Clinically relevant aminoglycoside resistance of Czech A. baumannii strains is significantly associated with the genes encoding enzymatic modification of kanamycin, gentamicin, amikacin and tobramycin. These genes can spread horizontally and emerge in different combinations leading to high-level resistance to multiple aminoglycosides. The AdeABC pump is likely to play a role in the intermediate susceptibility to netilmicin but further study is needed in this regard.