Most aminoglycoside resistance in Acinetobacter spp. involves production of aminoglycoside-modifying enzymes. Previous studies have shown that the genes encoding these enzymes can be present on plasmids, transposons or within integron-type structures. To determine whether particular mechanisms of aminoglycoside resistance have developed in strains from specific geographical locations (with subsequent clonal spread), or whether common mechanisms have been acquired by genotypically distinct clinical isolates of Acinetobacter spp. throughout the world, a genotypically heterogeneous collection of 24 multiresistant clinical isolates of Acinetobacter spp. from 15 hospitals in 11 countries worldwide was studied. All were resistant to two or more aminoglycoside antibiotics. The full aminoglycoside resistance profile was determined for each isolate, allowing a putative enzyme content to be inferred, with subsequent confirmation of enzyme content and genetic location by polymerase chain reaction (PCR) and hybridisation techniques. All produced at least one aminoglycoside-modifying enzyme, most commonly AAC(3)-I and ANT(3'')-I in various combinations. Other enzymes found were AAC(3)-II, AAC(6')-I, ANT(2''), APH(3')-I and APH(3')-VI. None was confined to strains from a particular geographical area. Nine isolates transferred resistance mediated by AAC(3)-I, ANT(2'')-I, APH(3')-I or APH(3)'-VI by conjugation to a sensitive strain of A. baumannii, but most resistance was non-transferable. PCR mapping revealed an integron location in six isolates for the aac(3)-Ia gene and in three isolates for the ant(3'')-Ia gene. Overall, the study demonstrated that similar aminoglycoside-modifying enzymes are found in unrelated isolates of Acinetobacter spp., and that particular genes are not restricted to specific areas of the world. The demonstration of certain genes on plasmids and integrons emphasises the probable importance of these structures in the dissemination of certain types of aminoglycoside resistance in Acinetobacter spp.