Myomycin is an unusual pseudodisaccharide antibiotic with a β-lysyl oligopeptide ester side chain that has structural similarities with kasugamycin, streptomycin and streptothricin. We show that the mode of action of myomycin in vivo and in vitro closely resembles that of streptomycin; in addition, spontaneous myomycin-resistant mutants of Escherichia coli are essentially indistinguishable from streptomycin-resistant mutants at the rRNA and r-protein level. Myomycin is not a substrate for the known streptomycin-modifying enzymes and could be useful in the characterization of natural streptomycin-resistant isolates and in counter-selecting against the presence of streptomycin-modifying enzymes. The relationship between structure and inhibition of protein synthesis has been examined for a series of derivatives of myomycin.