Aminoglycoside 3'-phosphotransferases I and II in three strains of Pseudomonas aeruginosa were studied in comparison with those in two strains of R factor-carrying Escherichia coli. The strain TI-13 of P. aeruginosa produced the former and strain H-9 the latter. Strain B-13 produced the both enzymes. The 3'-phosphotransferases of type I in P. aeruginosa TI-13, B-13 and E. coli K12 J5 R11-2 were different from each other in chromatographic behavior, molecular weight, pH optimum, and Ii. The 3'-phosphotransferase of type II in P. aeruginosa H-9 and E. coli JR66/W677 showed the same behavior.