When cultured in the presence of 600 mM D-mannitol, Escherichia coli K-12 cells synthesized two novel phospholipids. The identities of these compounds are postulated to be phosphatidylmannitol and diphosphatidylmannitol, the sugar alcohol analogs of phosphatidylglycerol and cardiolipin, respectively. The nonacylated glycerol moieties of the normal acidic phospholipids were substituted by D-mannitol. The formation of the analogs was significantly enhanced when strains harboring the pss-1 allele, a temperature-sensitive mutation in phosphatidylserine synthase (Ohta and Shibuya, J. Bacteriol. 132:434-443, 1977), were grown at 42 degrees C, and the accumulation of the analogs was maximum in late stationary phase; more than 90% of the total cellular lipids were these novel phospholipids. Strains with a defective cardiolipin synthase (Pluschke et al., J. Biol. Chem. 253:5048-5055, 1978) failed to form the analog lipids, whereas cells with increased cardiolipin synthase activity due to the presence of a pBR322-derived recombinant plasmid containing the structural gene for cardiolipin synthase produced more mannitol lipids than wild-type strains. These observations and the structures of the analog lipids indicated that cardiolipin synthase participates in the formation of these novel phospholipids. We suggest that reversible alcoholysis and condensation, in addition to low substrate specificity of the enzyme, are the mechanisms involved in this process. Addition to the medium of other straight-chain alditols, D-arabitol, ribitol, xylitol, erythritol, and L-threitol also yielded pairs of novel phospholipids, whereas sorbitol or galactitol produced only one analog in small quantities. These acidic phospholipid analogs have not been reported in any living system. They should be useful in the study of structure-function relationships of phospholipids and in manipulating the structures of various membrane systems.