Membrane fluidity and thermotropic phase behavior in the wild-type and plasmalogen-deficient strains of Megasphaera elsdenii have been studied by means of diphenylhexatiene steady state fluorescence anisotropy in isolated membranes, and by 31P-NMR and X-ray diffraction of the isolated phospholipids. Compared to the wild-type plasmalogen content of greater than 75%, plasmalogen-deficient strains had less than 5% plasmalogen, consisting largely of phosphatidylethanolamine and phosphatidylserine. Steady state fluorescence anisotropy measurements yielded an order parameter which was 6% lower in the plasmalogen-deficient membranes from 10 degrees to 40 degrees C, indicating higher membrane lipid mobilities. Both 31P-NMR and X-ray diffraction revealed the formation of a hexagonal phase in the lipids from the wild-type strain starting above 30 degrees C. In general the transition was not complete by 80 degrees C. In contrast, phospholipids from plasmalogen-deficient strains appeared to form a relatively stable lamellar phase.