Netilmicin, a semisynthetic derivative of sisomicin, was tested in vitro against 600 clinical bacterial isolates. At a concentration of 1.56 mug/ml, over 90% of gram-negative bacilli were inhibited. Netilmicin was substantially more active against isolates of Serratia marcescens and Enterobacter spp. than gentamicin, sisomicin, tobramycin, or amikacin. Isolates of Staphylococcus aureus (both penicillin G susceptible and resistant) were quite susceptible to netilmicin. Most isolates of Klebsiella spp. and Serratia spp. and some of the isolates of Pseudomonas aeruginosa that were resistant to gentamicin proved to be susceptible to netilmicin.