The aminoglycoside-aminocyclitol antibiotics constitute one of the antibacterial agent families with greater activity upon aerobic Gram-negative bacilli. These compounds are formed by the combination of one amino-cyclic alcohol (aminocyclitol) and aminosaccharides (aminoglycosides) linked by glycosidic bonds. The strong bactericidal activity exhibited for these compounds is not only explained by their ability to inhibit the protein synthesis, but also by pleiotropic effect altering the permeability of cytoplasmatic membrane. The penetration of these antibiotics to the bacterial cells is mediated by three well defined phases, being the two latest dependant of the proton-motive force. This fact explains that this kind of compounds have no antibacterial activity upon anaerobic bacteria. Bacterial resistance to aminoglycosides is mainly due to aminoglycoside-modifying enzymes (AME), which are commonly encoded by extrachromosomal genetic elements as are plasmids and transposons. Nevertheless, new mechanisms of resistance and genetic elements participating in the resistance to these compounds have been identified. Thus, recently the methylation of the 16S rRNA binding the aminoglycosides has been described. On the other hand, the gene cassettes acquire an increasing importance, because they can host a varied of families of antibiotic resistance genes, including the aminoglycoside-aminocyclitols. These gene cassettes are associated to integrons, which are able to integrate and express these antibiotic resistance determinants.