Protein E, a 91-residue membrane protein of phiX174, causes lysis of the host in a growth-dependent manner reminiscent of cell wall antibiotics, suggesting E acts by inhibiting peptidoglycan synthesis. In a search for the cellular target of E, we previously have isolated recessive mutations in the host gene slyD (sensitivity to lysis) that block the lytic effects of E. The role of slyD, which encodes a FK506 binding protein-type peptidyl-prolyl cis-trans isomerase, is not fully understood. However, E mutants referred to as Epos (plates on slyD) lack a slyD requirement, indicating that slyD is not crucial for lysis. To identify the gene encoding the cellular target, we selected for survivors of Epos. In this study, we describe the isolation of dominant mutations in the essential host gene mraY that result in a general lysis-defective phenotype. mraY encodes translocase I, which catalyzes the formation of the first lipid-linked intermediate in cell wall biosynthesis. The isolation of these lysis-defective mutants supports a model in which translocase I is the cellular target of E and that inhibition of cell wall synthesis is the mechanism of lysis.