Aims: The 2-deoxystreptamine-containing aminoglycoside antibiotics (AGAs) constitute the largest subgroup of the aminoglycosides. Neomycin (NEO) and lividomycin (LIV) are both representatives of the pseudo-tetrasaccharide group among the NEO-type AGAs. While NEO contains a 6'-NH(2) group, the 6'-position remains unmodified in LIV. The aim of the study was to characterize the substrate specificities of the enzymes involved in the C-6'- and C-6'''-modification in order to explain the different amination patterns. Methods and Results: We overproduced and purified the enzymes NeoQ (bifunctional 6'- and 6'''-oxidoreductase) and NeoB (bifunctional 6'- and-6'''-aminotransferase), which had been analysed before (Huang et al. 2007), and compared the enzymatic properties with the corresponding enzymes LivQ (postulated 6'''-oxidoreductase, 72% identity to NeoQ) and LivB (postulated 6'''-aminotransferase, 71% identity to NeoB) from the LIV pathway. By applying a newly established photometric assay, we proved that LivQ oxidized only pseudotetrasaccharidic substrates at the 6'''-position. In contrast, NeoQ accepted also the pseudodisaccharidic paromamine as a substrate and oxidized the 6'-and 6'''-positions on two different precursors of NEO. The aminotransferases LivB and NeoB both transfer NH(2) groups to the 6-position in the precursor 6'-oxo-paromamine and to the 6'''-position of 6'''-oxoneomycin C. Conclusions: The difference in the modification pattern of NEO and LIV at their 6'-positions is based only on the difference in the substrate specificities of the oxidoreductases LivQ and NeoQ, respectively. The aminotransferases LivB and NeoB share identical biochemical properties, and both are capable to transaminate the 6' and also the 6'''-position of the tested AGAs. Significance and Impact of the Study: Our data provide information to understand the structural variations in aminoglycosides and may be helpful to interpret variations in other natural product bisoynthesis pathways.