Two β-lactam antibiotics, cephalexin and FL 1060, which alone cause characteristic, distinct morphologic responses in Escherichia coli, gave rise, when used in combination, to the classical penicillin-induced response of spheroplast formation through discrete lesions in the cell wall. These results suggest that β-lactam antibiotics generally have two sites of action in E. coli and that cephalexin and FL 1060 each lack one of the normal functions of the β-lactam structure.