The antibiotic moenomycin is a valuable biochemical tool for studying the metabolism of peptidoglycan and the autolytic system in Escherichia coli, since as a specific inhibitor of peptidoglycan polymerases it can efficiently promote cell lysis. In liquid media the bacteriolytic effect on E. coli K12 was dependent on the concentration of moenomycin, on growth phase and on growth rate. Before lysis cells underwent major morphological alterations. In sucrose-containing medium complete transformation to osmotically sensitive spheroplasts was easily achieved by addition of moenomycin. The minimum inhibitory concentration of the antibiotic varied with the strain of E. coli and was highly dependent on the growth medium. A tritiated derivative of moenomycin, [3H]decahydromoenomycin A, was prepared and found to have the same inhibiting efficiency. Its binding to E. coli membranes and membrane proteins was investigated. The absence of irreversible binding suggested that moenomycin might be a competitive inhibitor of the peptidoglycan polymerases. Spontaneous moenomycin resistant variants were isolated at a frequency of about 10(-9).