The characteristic features of determination of the biological activity of neomycin and its separate components A, B and C were studied. Different sensitivity of 5 test microbes to neomycins A, B and C was observed. Bac. subtilis ATCC 6633 and Bac. cereus, var. myc. 537 were the most sensitive to neomycin A. Sar. lutea were not sensitive to neomycins A and C. The diffusion capacity of neomycin A was shown to be different from that of neomycins B and C. Even minute admixtures of neomycin A in the preparations of neomycin significantly changes the dose-response curves and affected the results of the biological activity determination.