A soluble, stable, L-glutamine:aminocyclitol aminotransferase has been purified 100-fold from mature mycelia of the gentamicin producer Micromonospora purpurea ATCC 15835. An absorbance maximum at 415 nm indicated the presence of pyridoxal phosphate in the enzyme; its involvement in the reaction, and hence its location at the active site, was indicated by abolition of the absorbance maximum upon addition of amino donors, and its restoration upon addition of amino acceptors. In addition, inhibition of the aminotransferase activity by carbonyl reagents was partially reversed by pyridoxal phosphate but not pyridoxal, confirming the importance of pyridoxal phosphate for reactivity. The keto-acid reaction product of transaminations with L-glutamine was identified as 2-ketoglutaramate, which exists in equilibrium with the inactive lactam, 2-hydroxy-5-oxoproline. Substrate specificity studies indicated that this aminotransferase is distinct from the commonly encountered aminotransferases since it did not readily catalyze transaminations with L-glutamate, L-aspartate, L-alanine, 2-ketoglutarate, oxaloacetate, or pyruvate. Active as amino donors were N('3)-methyl-2-deoxystreptamine, 2-deoxystreptamine, streptamine, scyllo-inosamine, and L-glutamine. Active as amino acceptors were scyllo-inosose, D,L-epi-inosose, and 2-ketoglutaramate. This aminotransferase preparation was able to catalyze transaminations which are compatible with both aminotransferase steps in the 2-deoxystreptamine biosynthetic pathway. Appearance of the enzyme during growth of M. purpurea, plus the substrate specificity, indicated that this aminotransferase is an idiophase enzyme, specific for 2-deoxystreptamine biosynthesis. A 2-deoxystreptamine-less idiotroph, M. purpurea ATCC 31119, was examined and found to be an excellent alternative source for the purification of this aminotransferase. The L-glutamine:aminocyclitol aminotransferase from crude extracts of idiophase cells of the neomycin producer, Streptomyces fradiae ATCC 10745, exhibited a substrate specificity very similar to that of the aminotransferase from M. purpurea. In addition, a 2-ketoglutaramate-hydrolyzing amidase activity was identified in both M. purpurea and S. fradiae. A functional relationship may exist between the L-glutamine:aminocyclitol aminotransferases and the amidases, as has been speculated for the L-glutamine:keto-acid aminotransferase and omega-amidase from rat tissues.