A number of bacterial strains, each possessing a different aminoglycoside-modifying enzyme, were examined for susceptibility to sisomicin and gentamicin B and the semisynthetic derivatives 5-epi-sisomicin and 5-epi-gentamicin B. Although strains possessing AAC (6') or APH(3') enzymes were equally resistant to the 5-epi-compounds, those possessing AAC(3)-I, ANT(2"), or AAC(2') enzymes were much more sensitive to the 5-epi derivatives. Analysis of partially purified aminoglycoside-modifying enzymes from the strains showed that the 5-epi compounds were substrates even for those enzymes found in susceptible strains [AAC(3)-I, ANT(2"), and AAC(2')]. However, a more detailed study of the enzymes showed that they had much increased Km values for the 5-epi derivatives; the 5-epi compounds were much less effectively modified than the parent antibiotics. This confirms and extends the notion that enzymatic modification of aminoglycosides is not in itself sufficient to confer resistance to the drugs, but also that the modification must be efficient, as reflected in the Km values.