A purified preparation of N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28), a murein hydrolase from Escherichia coli, was found to lose its activity during incubation in the presence of bacterial phospholipid suspensions. Whether it was co-dispersed with the phospholipids or added to sonicated phospholipid suspension, the enzyme was inhibited (or inactivated) from the first minutes of incubation at 37 degree C. As phosphatidylglycerol/cardiolipin ratio of the phospholipid suspension as increased (all other things being equal), a further decrease of amidase activity was observed. The highest losses of activity were found after co-dispersion of the enzyme and the substrate together with the phospholipids, the resulting suspension being formed of larger multilayered vesicles, as revealed by electron microscopy. In these conditions, the effect on enzyme activity was only partially accounted for by the proportion of the enzyme that was entrapped in the vesicles. The entrapment capacity of the enzyme (using a 35S-labelled enzyme preparation) and of the substrate (3H-labelled) by the multilamellar phospholipidic vesicles did not significantly change as a function of their relative content of phosphatidylglycerol and cardiolipin. The possible physiological meaning of the results is discussed is connection with our previous data and with other works related to membranous phospholipid distribution and to septum formation control in bacteria.