The role of phospholipid synthesis in peptidoglycan metabolism during growth of Escherichia coli was determined. The inhibition of phospholipid synthesis, achieved by inhibiting fatty acid synthesis with cerulenin or by glycerol deprivation of gpsA mutant strains, resulted in the concomitant inhibition of peptidoglycan synthesis. These effects on peptidoglycan synthesis were relatively specific in that the treatments did not cause a general inhibition of macromolecular synthesis. Furthermore, the inhibition of phospholipid synthesis also resulted in the rapid development of penicillin tolerance. It was unlikely that penicillin tolerance in these cases were simply due to the inhibition of growth caused by cerulenin treatment or glycerol deprivation because treatments with more effective growth inhibitors, e.g. chloramphenicol or norfloxacin, did not confer penicillin tolerance. Penicillin tolerance was shown to be a direct consequence of the inhibition of phospholipid synthesis and not due to the possible accumulation of guanosine-3',5'-bispyrophosphate (ppGpp), the starvation stress signal molecule known to be responsible for the development of penicillin tolerance in amino-acid-deprived bacteria. Therefore, peptidoglycan metabolism is coupled to phospholipid synthesis during growth of E. coli, and this may represent an important means to ensure the coordination of cell envelope synthesis in growing bacteria.