Sixteen amikacin-resistant clinical Acinetobacter baumannii isolates from nine different hospitals in Spain were investigated to determine whether the high incidence of amikacin-resistant A. baumannii was due to the dissemination of an amikacin-resistant strain or to the spread of an amikacin resistance gene. The epidemiological relationship studied by repetitive extragenic palindromic PCR and low-frequency restriction analysis of chromosomal DNA showed that the same clone was isolated in eight of nine hospitals, although other clones were also found. The strains were studied for the presence of the aph(3')-VIa and aac(6')-I genes, which encode enzymes which inactivate amikacin, by PCR. All 16 clinical isolates had positive PCRs with primers specific for the amplification of the aph(3')-VIa gene, whereas none had a positive reaction for the amplification of the aac(6')-I gene. Therefore, the high incidence of amikacin resistance among clinical A. baumannii isolates in Spain was mainly due to an epidemic strain, although the spread of the aph(3')-VI gene cannot be ruled out.