The cells of an L-form strain of Streptomyces hygroscopicus have been grown for 20 years without a cell wall. Their cytoplasmic membranes have high stability and an unusual structural polymorphism. To clarify the importance of the lipid components for these membrane properties, a comparative analysis has been carried out with purified membranes of L-form cells, of parent vegetative hyphal cells (N-form cells), and of protoplasts derived from the latter. The phospholipid classes and fatty acids were determined by thin-layer chromatography (TLC), two-dimensional TLC, high-performance liquid chromatography, gas chromatography, and mass spectrometry. The qualitative compositions of cardiolipin (CL), lyso-cardiolipin (LCL), phosphatidylethanolamine (PE1 and PE2), lyso-phosphatidylethanolamine (LPE), phosphatidylinositolmannoside (PIM), phosphatidic acid (PA), dilyso-cardiolipin-phosphatidylinositol (DLCL-PI), and the 13 main fatty acids were the same in the three membrane types. However, significant quantitative differences were observed in the L-form membrane. They consist of a three-to fourfold-higher content of total, extractable lipids, 20% more phospholipids, an increased content of CL and PIM, and a reduced amount of the component DLCL-PI. Furthermore, the L-form membrane is characterized by a higher content of branched anteiso 15:0 and anteiso 17:0 fatty acids compared to that of the membranes of the walled vegetative cells. These fatty acids have lower melting points than their straight and iso-branched counterparts and make the membrane more fluid. The phospholipid composition of the protoplast membrane differs quantitatively from that of the N form and the L form. Whereas the phospholipid classes are mostly similar to that of the N form, the fatty acid pattern tends to be closer to that of the L-form membrane. The membranes of both the L-form cells and the protoplasts need to be more fluid because of their spherical cell shape and higher degree of curvature compared with N-form membranes.Streptomycetes are bacteria characterized by a high degree of morphological and biochemical differentiation. Streptomyces hygroscopicus grows as branched hyphae, and it is able to form an aerial mycelium and spores. It produces several secondary metabolites, especially the antibiotic turimycin. The vegetative cells are 3-to 20-m-long hyphal units surrounded by a cytoplasmic membrane and a typical 30-nm-thick cell wall. From this species, we could isolate an L-form strain, which is able to propagate as cell wall-less, spherical, or pleomorphic cells on agar media as well as in liquid media under shaking conditions (2, 12).The stable protoplast type L-form strain is the result of an adaptive process. It has lost, irreversibly, the capability to resynthesize cell wall structures, and it represents a genetically stable mutant showing extreme pleomorphic alterations. These involve cell and colony morphology, growth behavior, biochemical activities, and an incapability to form spores and secondary metabolites (12, 13). In particu...