The aminoglycoside phosphotransferase of Pseudomonas aeruginosa 21-75 was purified by affinity chromatography using dibekacin-Sephadex 4B or lividomycin A-Sepharose 4B followed by DEAE Sephadex A-50 chromatography. It had activities of both the known aminoglycoside 3'-phosphotransferases I and II, and transferred phosphate from ATP to the 3'-hydroxyl group of kanamycin A, ribostamycin and butirosin A and 5-hydroxyl group of lividomycin A. This enzyme was designated aminoglycoside 3'-phosphotransferase III. It showed strong substrate inhibition by kanamycin A and ribostamycin when their concentration exceeded 6 muM. Purification and characterization of this enzyme are reported.