Using insertional inactivation of the different genes of the dlt operon in Bacillus subtilis, we searched for metabolic and morphological changes caused by D-alanine ester deprivation of lipoteichoic acid and wall teichoic acid. There were no alterations of cell growth, basic metabolism, cellular content of phosphorus-containing compounds, ultrastructure, cell separation, and surface charge. The only alteration observed was an enhancement of endogenous and beta-lactam-induced cell lysis. Since this enhancement is doubtless correlated with the D-alanine ester deprivation of the teichoic acids, the present view based on in vitro experiments, that negatively charged LTA is inhibitory to autolysins, may be questioned. We propose that negatively charged lipoteichoic acid and/or wall teichoic acid serve in vivo to fix the cationic autolysins within the cell wall-membrane complex by electrostatic interaction. Positively charged D-alanine ester substituents decrease the binding capacity of the teichoic acids for autolysins by charge compensation.