Description: Recently, iso -diabolic acid (13,16-dimethyl octacosanedioic acid) has been identified as a major membrane-spanning lipid of subdivisions 1 and 3 of the Acidobacteria , a highly diverse phylum within the Bacteria . This finding pointed to the Acidobacteria as a potential source for the bacterial glycerol dialkyl glycerol tetraethers that occur ubiquitously in peat, soil, lakes, and hot springs. Here, we examined the lipid composition of seven phylogenetically divergent strains of subdivision 4 of the Acidobacteria , a bacterial group that is commonly encountered in soil. Acid hydrolysis of total cell material released iso -diabolic acid derivatives in substantial quantities (11 to 48% of all fatty acids). In contrast to subdivisions 1 and 3 of the Acidobacteria , 6 out of the 7 species of subdivision 4 (excepting " Candidatus Chloracidobacterium thermophilum") contained iso -diabolic acid ether bound to a glycerol in larger fractional abundance than iso -diabolic acid itself. This is in agreement with the analysis of intact polar lipids (IPLs) by high-performance liquid chromatography-mass spectrometry (HPLC-MS), which showed the dominance of mixed ether-ester glycerides. iso -Diabolic acid-containing IPLs were not identified, because these IPLs are not released with a Bligh-Dyer extraction, as observed before when studying lipid compositions of subdivisions 1 and 3 of the Acidobacteria . The presence of ether bonds in the membrane lipids does not seem to be an adaptation to temperature, because the five mesophilic isolates contained a larger amount of ether lipids than the thermophile " Ca . Chloracidobacterium thermophilum." Furthermore, experiments with Pyrinomonas methylaliphatogenes did not reveal a major influence of growth temperature over the 50 to 69°C range.