Gentamicin-resistant strains of Pseudomonas species have been examined for the presence of inactivating enzymes. Of 44 isolates of P. aeruginosa resistant to gentamicin only 10 produced inactivating enzymes. Five strains produced aminoglycoside 3-N-acetyltransferase I (AAC(3)I) and 5 strains produced aminoglycoside 2″-O-nucleotidyltransferase. Four strains produced an enzyme, aminoglycoside 6′-N-acetyltransferase, that does not account for their resistance to gentamicin. The remaining strains did not produce inactivating enzymes but showed increased resistance to all aminoglycosides including amikacin and tobramycin. Of 42 gentamicin-resistant strains of other species of Pseudomonas only one, a strain of P. pseudoalcaligenes, produced a gentamicin-inactivating enzyme, AAC(3)I. A number of gentamicin-sensitive and resistant strains of P. aeruginosa produced streptomycin phosphotransferase or streptomycin adenylyltransferase but these enzymes were not detected in other species of Pseudomonas. Aminoglycoside 3′-O-phosphotransferase was found in all strains of P. aeruginosa that were tested, and in one strain of P. pseudoalcaligenes.