Among the eight strains of Listeria monocytogenes tested for lysozyme sensitivity, two were resistant to lysozyme but became sensitive after lipase pretreatment. Among the other six, one was very sensitive to lipase and another one was extremely susceptible to lysozyme. Stable protoplasts were formed from the lysozyme-resistant strain (42) by lipase and lysozyme treatment, which completely digested the cell wall. The cell wall (uranyl acetate-lead stained) was of a thick triplelayered profile, with the intermediate layer of low density. Lipase treatment for a short time (60 min) did not cause any alteration in structure, but prolonged treatment (180 min) caused extensive digestion of the plasma membrane and the cell wall, liberating cytoplasmic material. When the cells were treated with either lipase or lysozyme, a small number of protoplasts were extruded through the partly digested or weakened transverse cell wall, leaving an almost intact cell wall ghost. There were small vesicular structures in the interspace between cell wall and plasma membrane. Mesosomes of varied organization were prominent in electron micrographs, both in sections and in negatively stained preparations. These were largely everted during protoplasting in the form of tubules and as small peripheral buds; a few small vesicles also remained as intrusive structures, some of which were very unusual because they appeared to be enclosed by the inner layer of plasma membrane alone. Lysis of the protoplasts by dilution of the sucrose, while maintaining a constant ionic environment, liberated many small vesicular structures and fibrillar nuclear material. Listeria monocytogenes is a small gram-positive bacillus which possesses a fine structure with an unusual organization (4). Typical dimensions are 0.4 to 0.5 by 0.5 to 2.0 At, but the morphology and Gram reaction may vary under different growth and nutritional conditions (18). The cells are surrounded by a thick multilayered cell wall (7) and a relatively thick plasma membrane (10). The latter has five distinct layers (4). The intracytoplasmic membranous structures, termed "mesosome" by Fitz-James (5) and "plasmalemmasome" by Edwards and Stevens (4), have varied organization and extensive distribution (4, 7, 10). The production of protoplasts of L. monocytogenes was originally undertaken with the aim of determining the location in the cell of the monocyte-producing agent (MPA), which was reported I Post-doctoral Fellow, Medical Research Council of Canada 1964-1966. by Stanley (19) to be present in lipid extracts of the organism. Marked variations in lysozyme sensitivity were encountered in different strains, and this paper describes methods used for protoplast formation and interesting aspects of the ultrastructure in relation to formation of the protoplast. MATERIALS AND METHODS Maintenance and cultivation of organism. Various strains of Listeria monocytogenes from the collection of the late E. G. D. Murray were used in this study. Stock cultures were maintained at room temperature on tryptose-ag...