The mechanism of resistance of the gentamicin-producing organism Micromonospora purpurea was analyzed. Determination of minimal inhibitory concentrations revealed high resistance to the 4,6-substituted deoxystreptamine aminoglycosides amikacin, gentamicin, kanamycin, netilmicin, sisomicin, and tobramycin and also to lividomycin A and hygromycin B, but susceptibility to streptomycin, dihydrostreptomycin, paromomycin, and neomycin during all phases of the growth cycle. The nonproducing, closely related Micromonospora melanosporea was susceptible to these compounds. In agreement with results from previous studies (R. Benveniste and J. Davies, Proc. Natl. Acad. Sci. U.S.A. 70:2276-2280, 1973), extracts from M. purpurea showed no activity of enzymes specifically modifying gentamicin. 70S ribosomes from M. purpurea but not from M. melanosporea were resistant to inhibition by gentamicin, kanamycin, tobramycin, and lividomycin in a polyuridylic acid-dependent polyphenylalanine synthesis system and susceptible to those compounds which were inhibitory in vivo. The former antibiotics were also unable to induce misreading. Subunit exchange experiments between M. purpurea and M. melanosporea showed that the main site for inhibition and induction of misreading is the 30S subunit (up to gentamicin concentrations of 10 micrograms/ml).