Cells of the photosynthetic bacterium Rhodobacter sphaeroides grown under phosphate-limiting conditions accumulated nonphosphorous glycolipids and lipids carrying head groups derived from amino acids. Concomitantly, the relative amount of phosphoglycerolipids decreased from 90 to 22 mol% of total polar lipids in the membranes. Two lipids, not detectable in cells grown under standard conditions, were synthesized during phosphate-limited growth. Fast atom bombardment mass spectroscopy, exact mass measurements, 1H NMR spectroscopy, sugar composition analysis, and methylation analysis of the predominant glycolipid led to the identification of the novel compound 1,2-di-O-acyl-3-O-[alpha-D-glucopyranosyl-(1-->4)-O-beta-D-galactopyr anosyl]glycerol. The second lipid was identified as the betaine lipid 1,2-di-O-acyl-[4'-(N,N,N-trimethyl)-homoserine]glycerol by cochromatography employing an authentic standard from Chlamydomonas reinhardtii, fast atom bombardment mass spectroscopy, exact mass measurements, and 1H NMR spectroscopy. Prior to this observation, the occurrence of this lipid was thought to be restricted to lower plants and algae. Apparently, these newly synthesized nonphosphorous lipids, in addition to the sulfo- and the ornithine lipid also found in R. sphaeroides grown under optimal conditions, take over the role of phosphoglycerolipids in phosphate-deprived cells.