Two independent mutants of Escherichia coli lacking murein-lipoprotein have been found. One mutant whose mutation was named lpo was subjected to detailed analyses. The absence of both bound and unbound lipoproteins was shown by electrophoretic analysis of 14C-arginine labelled membrane proteins of the mutant. Nor was serologically cross-reacting material detected in the mutant by the Ouchterlony-method. Sequestering magnesium from mutant cell suspensions by ethylenediaminetetraacetic acid caused cell lysis, which was prevented in the presence of 0.5 M sucrose. Incubation in culture media at a very low level of magnesium resulted in the formation of blebs in the mutant. Examination of mutant cells by electron microscopy showed that the outer membrane of the mutant was uneven with small irregular protuberances, some of which pinched off forming vesicles of various sizes. Phosphotungstate used for negative-staining penetrated into the periplasmic space of the mutant cells. The mutants leaked a considerable fraction of their periplasmic enzymes. These physiological and morphological alterations in the lipoproteinless mutant suggest that murein-lipoprotein helps to maintain the outer envelope structure by connecting the outer membrane with murein so that the outer membrane may fulfil its physiological functions as a barrier to the environment.