SUMMARY: Escherichia coli NCTC86 organisms with impaired walls were prepared by three methods and the effect of antibacterial agents on their growth studied. The growth of penicillin spheroplasts was masked by the overgrowth of unaltered cells in the culture; the EDTA-lysozyme spheroplasts themselves were non-viable. The growth of penicillin spheroplasts was not affected by cell-wall inhibitors and ampicillin suppressed the overgrowth of unaltered cells. The sensitivity of penicillin spheroplasts and parent cells to inhibition by a range of agents was similar. EDTA treatment enhanced the susceptibility of E. coli NCTC86 and other strains of Gram-negative species to several antibiotics, particularly erythromycin. Polyacetic acid chelating agents related to EDTA and some new amides derived from glycine, alanine, phenylalanine or methionine also potentiated erythromycin in vitro. Erythromycin showed some activity in protecting mice against infection by EDTA-treated E. coli NCTC86. The antibiotic did not protect against infection by the untreated bacteria and its activity was not greatly enhanced by simultaneous administration of EDTA or the amide derivatives.