The polar lipids from group B Streptococci have been isolated. Unless overlapping fractions are worked up on each step molecular species can be lost resulting in a fatty acid composition different from the original one. 2. The lipids were shown to be 1(3),2-diacyl-3(1)-O-alpha-D-glucopyranosyl-sn-glycerol, 1(3),2-diacyl-3(1)-O-[alpha--D-glucopyranosyl-(1,2)-O-alpha-D-glucopyranosyl]-sn-glycerol, 1,2-diacyl-sn-glycero-3-phospho-1'-sn-glycerol, lysylphosphatidylglycerol and 1'-3'-bis(1,2-diacyl-sn-glycero-3-phospho-glycerol. The sterochemical configuration of the phospholipids was achieved by combinations of chemical degradations. 3. The fatty acid composition of group B Streptococci lipids is qualitatively the same as in other Streptococci, but differs by a high content of stearic acid and by a low degree of unsaturation and cyclopropanization. 4. The forementioned polar lipids as well as the glucosyldiphsphatidylglycerol,, which is also found in this organism, showed a very similar composition and the same non-radom positional distribution of fatty acids. Apart from short chain fatty acids the positioning differentiates between saturated and unsaturated fatty acids: short chain and unsaturated fatty acids are accumulated at Position 2, saturated acids are preferentially linked to Position 1. 5. The uniform fatty acid make-up in all polar lipids favours the hypothesis that their diacylglycerol portions are derived from a common phosphatidic acid precursor with negligible postsynthetic rearrangements of the constituent fatty acids.