Summary: The phospholipid ester-linked fatty acids were examined in four Desulfobacter strains (2ac9, AcBa, 3ac10 and 4ac11), a Desulfobacter-like ‘fat vibrio’ (AcKo) and Desulfotomaculum acetoxidans (5575), which are all sulphate-reducing bacteria that oxidize acetate. A thermophilic sulphate reducer, Desulfovibrio thermophilus, and two sulphur-reducing bacteria, Desulfuromonas acetoxidans (11070) and a Campylobacter-like spirillum (5175), were also studied. The Desulfobacter spp. were characterized by significant quantities of 10-methylhexadecanoic acid. Other 10-methyl fatty acids were also detected in Desulfobacter spp. No 10-methyl fatty acids were detected in the other organisms examined, supporting the use of 10-methylhexadecanoic acid as a biomarker for Desulfobacter. High levels of cyclopropyl fatty acids, including two isomers of both methylenehexadecanoic (cy17:0) and methyleneheptadecanoic (cy18:0) acids, were also characteristic of Desulfobacter spp. The influence of the volatile fatty acids (VFA) propionate, isobutyrate, isovalerate and 2-methylbutyrate on the lipid fatty acid distribution was studied with two Desulfobacter strains (2ac9, AcBa) and Desulfotomaculum acetoxidans. Although these sulphate reducers cannot oxidize the VFA, their presence in the acetate growth medium caused a shift in the fatty acid distribution in favour of odd-numbered and branched chains by apparent direct incorporation into the fatty acids as chain initiators. The Desulfobacter strains were distinguished from other sulphide-forming bacteria by the percentage of unsaturated and the percentage of branched fatty acids.