In vitro tests for susceptibility to amikacin and to four other aminoglycoside antibiotics were carried out with strains of many bacterial species by use of an agar dilution method and an inocula replicator. In general, amikacin was as active as or more active against most of the organims than kanamycin, neomycin, and streptomycin; in particular, amikacin was active against strains resistant to one or more of these three antibiotics. Amikacin was more active than gentamicin against strains of Nocardia asteroides and Providencia stuartii and also against gentamicin-resistant strains of some other gram-negative bacilli, notably Serratia marcescens. However, gentamicin was more active than amikacin against most of the other gram-positive and gram-negative bacteria that were tested. In comparative tests of four media, minimal inhibitory concentrations MICs) were greater in tests with Mueller-Hinton agar, and generally somewhat lower in those with heart infusion agar, than in tests with trypticase soy agar and nutrient agar. Inocula of a 1:1,000 dilution of culture generally gave MICs lower than those obtained with undiluted cultures; the differences were small with enterococci, but they were greater with amikacin than with gentamicin in tests on strains of Klebsiella pneumoniae. These findings generally confirm those previously reported by others.