Eighty-seven isolates of Pseudomonas, Enterobacteriaceae, and Staphylococcus, chosen because of their resistance to other aminoglycosides, were tested for susceptibility to 5-episisomicin. Tests were performed in Mueller-Hinton agar and also, with 38 of these isolates, in Mueller-Hinton broth. Of Enterobacteriaceae, 85 and 95.5% were inhibited by 5 and 10 mug of 5-episisomicin per ml, respectively. Amikacin inhibited 74 and 91% of the strains at 10 and 20 mug/ml, respectively. Fifty-four percent of P. aeruginosa were inhibited by 5-episisomicin and amikacin. Eighty-three percent of S. aureus were inhibited by netilmicin and amikacin, whereas only 50% were inhibited by 5-episisomicin. Isolates resistant to 5-episisomicin were most often resistant to the other aminoglycosides and occurred in gram-negative bacilli that did not carry aminoglycoside-modifying enzymes. Five of 23 isolates that carried a 6'-N-acetyltransferase (AAC-6') and one of two that carried an aminoglycoside 3-acetyltransferase were resistant to and acetylate 5-episisomicin. Strains carrying other aminoglycoside-modifying enzymes were inhibited by 5-episisomicin. Thus, 5-episisomicin is a promising aminoglycoside not attacked by most aminoglycoside-modifying enzymes. Resistance will probably most often be based upon nonenzymatic mechanisms which will also affect other aminoglycosides.